Plant transformation

ABSTRACT

Plant cells and related systems, methods, and compositions for improving the capacity of the plant cells to regenerate embryogenic plant tissues, plant organs, and whole plants are provided. Such plant cells and related systems, methods, and compositions provide for increased expression of the endogenous morphoregulators BABYBOOM (ODP2) and/or WUSCHEL2 (WUS2).

REFERENCE TO RELATED APPLICATIONS

This is a continuation application of U.S. Ser. No. 16/844,438, filedApr. 9, 2020, which claims the benefit of priority to U.S. ProvisionalPatent Application No. 62/833,150, filed Apr. 12, 2019, which areincorporated herein by reference in their entireties.

INCORPORATION OF SEQUENCE LISTING

The sequence listing contained in the file named“63200_195755_SEQLISTING_ST25.txt”, which is 385,024 bytes measured inWindows, which was created on Apr. 9, 2020 and electronically filed viaEFS-Web on Apr. 9, 2020, is incorporated herein by reference in itsentirety.

BACKGROUND

To overcome recalcitrance of corn to genetic transformation, transgenesencoding morphoregulators BABYBOOM (ODP2) and/or WUSCHEL2 (WUS2) havebeen transiently expressed in corn cells to stimulate somaticembryogenesis (Lowe et al. 2016, Lowe et al. 2018). Such somaticembryogenesis promotes formation of Type II embryogenic callus fromwhich new shoots can be generated, allowing maize genotype independentgenetic transformation and making previously recalcitrant tissuesaccessible to transformation. The expression of BABYBOOM (ODP2) and/orWUSCHEL2 (WUS2) has also been shown to promote embryogenesis in sorghum(Mookkan et al. 2017), wheat, and a variety of other plants includingcotton (US Patent Appl. Pub. No. 20170342431; U.S. Pat. No. 7256322;also reviewed by Nagle et al., 2018).

It is not yet clear how BABYBOOM and WUSCHEL trigger this effect.BABYBOOM is known to induce somatic embryogenesis, the biology of whichis beginning to be understood (Horstman et al. 2017; Jha and Kumar,2019). WUSCHEL biology has been investigated primarily in Arabidopsis(Rodriguez et al. 2016; Schoof et al. 2000; Mayer et al. 1998; Laux etal. 1996). It has a major role in stem cell maintenance.

The genomic insertion of morphoregulator-encoding transgenes is not auniversal agricultural biotechnology solution, and also stipulatesmarket access consequences. Commercialization of transgenic plants, andtheir progeny, is restricted by country-specific regulations. Genomicinsertion of transgenes can facilitate the introduction of gene editingreagents, for example when coupled with transgenes encoding CRISPR-Cas9(Soda, Verma, and Giri 2017; W. Wang et al. 2018). Hence, transgenesoften need to be segregated away from edited progeny which can requiremultiple crosses and lengthen development/production timelines andcosts. Transgenes do enable the use of selectable markers which enrichfor edited tissue, greatly improving editing efficiency. Due to thisenrichment step transgenesis remains the primary method for producingedited plants, however several groups are experimenting with alternativeselection tools (Zhang et al. 2016; Hamada et al. 2018).

SUMMARY

Disclosed herein are plant cells wherein expression of an endogenousODP2 polypeptide and/or expression of an endogenous WUS2 polypeptide istransiently increased in comparison to the expression of the endogenousODP2 and/or the endogenous WUS2 polypeptides in a control plant cell,and wherein the plant cell can form a regenerable plant structure. Alsodisclosed are tissue cultures of such plant cells and related methodswherein the cells are used to obtain genetically edited or geneticallytransformed regenerable plant structures (e.g., a somatic embryo,embryogenic callus, somatic meristem, organogenic callus, a shoot, or ashoot further comprising roots) or plants. In certain embodiments, theexpression of the endogenous ODP2 polypeptide and/or the endogenous WUS2polypeptide is transiently increased in the plant cell with at least oneexogenous gene transcription agent that stimulates transcription of theendogenous ODP2 gene and/or with at least one exogenous genetranscription agent that stimulates transcription of the endogenous WUS2gene. Such plant cells include both monocot (e.g., maize, wheat,sorghum, and rice) plant cells and dicot plant cells (e.g., Brassicasp., cotton, and soybean). Also provided are maize plant cellscomprising at least one exogenous gene transcription agent thatstimulates transcription of the endogenous WUS2 gene, wherein expressionof the endogenous WUS2 polypeptide is increased in comparison to theexpression of the endogenous WUS2 polypeptide in a control maize plantcell, wherein the endogenous WUS2 polypeptide is encoded by anendogenous polynucleotide that is operably linked to an endogenous maizeWUS2 promoter of SEQ ID NO:4 or an allelic variant thereof, wherein theexogenous gene transcription agent(s) bind to DNA sequences in theendogenous maize WUS2 promoter corresponding to residues 100 to 225 ofSEQ ID NO:4, and wherein the maize plant cells can form a regenerablemaize plant structure.

Methods provided herein include methods of producing a regenerable plantstructure, comprising introducing into the plant cell at least oneexogenous gene transcription agent which transiently increasesexpression of an endogenous ODP2 polypeptide and/or at least oneexogenous gene transcription agent which increases expression of anendogenous WUS2 polypeptide, wherein the expression is increased incomparison to the expression of the endogenous ODP2 and/or theendogenous WUS2 polypeptides in a control plant cell; and culturing theplant cell to produce the regenerable plant structure. In certainembodiments of the methods, the exogenous gene transcription agentcomprises: (i) a domain or complex which binds to the promoter or 5′untranslated region (5′ UTR) of the endogenous ODP2 gene or to thepromoter or 5′ UTR of the endogenous WUS2 gene; and (ii) a transcriptionactivation domain, wherein the transcription activation domain isoperably linked or operably associated with the domain or complex. Suchmethods can be applied to plant cells that include both monocot (e.g.,maize, wheat, sorghum, and rice) plant cells and dicot plant cells(e.g., Brassica sp., cotton, and soybean). Also provided are methods ofproducing a regenerable maize plant structure, comprising: (i)introducing into a maize plant cell at least one exogenous genetranscription agent which transiently increases expression of anendogenous WUS2 polypeptide, wherein the expression is increased incomparison to the expression of the endogenous WUS2 polypeptide in acontrol maize plant cell, wherein the endogenous WUS2 polypeptide isencoded by an endogenous polynucleotide that is operably linked to anendogenous maize WUS2 promoter of SEQ ID NO:4 or an allelic variantthereof, and wherein the exogenous gene transcription agent(s) bind toDNA sequences in the endogenous maize WUS2 promoter corresponding toresidues 100 to 225 of SEQ ID NO:4; and, (ii) culturing the maize plantcell to produce a regenerable maize plant structure.

DETAILED DESCRIPTION

Unless otherwise stated, nucleic acid sequences in the text of thisspecification are given, when read from left to right, in the 5′ to 3′direction. Nucleic acid sequences may be provided as DNA or as RNA, asspecified; disclosure of one necessarily defines the other, as well asnecessarily defines the exact complements, as is known to one ofordinary skill in the art. Where a term is provided in the singular, theinventors also contemplate embodiments described by the plural of thatterm.

The phrase “allelic variant” as used herein refers to a polynucleotideor polypeptide sequence variant that occurs in a different strain,variety, or isolate of a given organism.

The term “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C;A and C; A and B; B and C; A (alone); B (alone); and C (alone).

As used herein, the terms “correspond,” “corresponding,” and the like,when used in the context of an nucleotide position, mutation, and/orsubstitution in any given polynucleotide (e.g., an allelic variant ofSEQ ID NO: 4) with respect to the reference polynucleotide sequence(e.g., SEQ ID NOs: 4, 101, 102, residues 130-210 of SEQ ID NO: 4) allrefer to the position of the polynucleotide residue in the givensequence that has identity to the residue in the reference nucleotidesequence when the given polynucleotide is aligned to the referencepolynucleotide sequence using a pairwise alignment algorithm (e.g.,CLUSTAL O 1.2.4 with default parameters).

As used herein, the terms “Cpf1” and “Cas12a” are used interchangeablyherein to refer to the same RNA directed nuclease.

The phrases “expression of an endogenous ODP2 polypeptide,” “expressionof an endogenous WUS2 polypeptide,” “expression of the endogenous ODP2polypeptide,” and “expression of the endogenous WUS2 polypeptide” referto the expression of an ODP2 polypeptide or WUS2 polypeptiderespectively encoded by an endogenous ODP2 gene or endogenous WUS2 genein a plant genome.

As used herein, the phrase “genome altering reagent’ refers to anymolecule or set of molecules that can result in either the site-specificor non-site specific insertion of an exogenous nucleic acid moleculeinto the genome or a site-specific or non-site specific insertion,deletion, and/or substitution of one or more nucleotide residues in thegenome. A genome altering reagent can comprise a transgene, a vectorcomprising a transgene, a genome editing molecule(s), and/orpolynucleotides encoding the genome editing molecule(s).

As used herein, the phrase “gene-editing” includes genome modificationby homology directed repair (HDR), base editing, and non-homologousend-joining (NHEJ) mechanisms. Such gene-editing includes embodimentswhere a site specific nuclease and a donor template are provided.

As used herein, an “exogenous” agent or molecule refers to any agent ormolecule from an external source that is provided to or introduced intoa system, composition, plant cell culture, reaction system, or plantcell. In certain embodiments, the exogenous agent (e.g., polynucleotide,protein, or compound) from the external source can be an agent that isalso found in a plant cell. In certain embodiments, the exogenous agent(e.g., polynucleotide, protein, or compound) from the external sourcecan be an agent that is heterologous to the plant cell.

As used herein, a “heterologous” agent or molecule refers: (i) to anyagent or molecule that is not found in a wild-type, untreated, ornaturally occurring composition or plant cell; and/or (ii) to apolynucleotide or peptide sequence located in, e.g., a genome or avector, in a context other than that in which the sequence occurs innature. For example, a promoter that is operably linked to a gene otherthan the gene that the promoter is operably linked to in nature is aheterologous promoter.

The phrase “improved plant cell regenerative potential” as used hereinrefers to the ability of a given plant cell to form a somatic embryo,embryogenic callus, a somatic meristem, organogenic callus, a shoot, ora shoot further comprising roots in comparison to a control plant cell.

As used herein, the terms “include,” “includes,” and “including” are tobe construed as at least having the features to which they refer whilenot excluding any additional unspecified features.

As used herein, the term “overproduced” where used herein with regardsto various agents refers to providing the agent in an amount that isincreased in comparison to the amount found in an untreated plant cellor plant.

As used herein, the phrase “plant cell” can refer either a plant cellhaving a plant cell wall or to a plant cell protoplast lacking a plantcell wall.

The term “polynucleotide” where used herein is a nucleic acid moleculecontaining two (2) or more nucleotide residues. Polynucleotides aregenerally described as single- or double-stranded. Where apolynucleotide contains double-stranded regions formed by intra- orintermolecular hybridization, the length of each double-stranded regionis conveniently described in terms of the number of base pairs.Embodiments of the systems, methods, and compositions provided hereincan employ or include: (i) one or more polynucleotides of 2 to 25residues in length, one or more polynucleotides of more than 26 residuesin length, or a mixture of both. Polynucleotides can comprise single- ordouble-stranded RNA, single- or double-stranded DNA, double-strandedDNA/RNA hybrids, chemically modified analogues thereof, or a mixturethereof. In certain embodiments, a polynucleotide can include acombination of ribonucleotides and deoxyribonucleotides (e.g., syntheticpolynucleotides consisting mainly of ribonucleotides but with one ormore terminal deoxyribonucleotides or synthetic polynucleotidesconsisting mainly of deoxyribonucleotides but with one or more terminaldideoxyribonucleotides), or can include non-canonical nucleotides suchas inosine, thiouridine, or pseudouridine. In certain embodiments, thepolynucleotide includes chemically modified nucleotides (see, e.g.,Verma and Eckstein (1998) Annu. Rev. Biochem., 67:99-134). Chemicallymodified nucleotides that can be used in the polynucleotides providedherein include: (i) phosphorothioate, phosphorodithioate, ormethylphosphonate internucleotide linkage modifications of thephosphodiester backbone; (ii) nucleosides comprising modified basesand/or modified sugars; and/or (iii) detectable labels including afluorescent moiety (e.g., fluorescein or rhodamine or a fluorescenceresonance energy transfer or FRET pair of chromophore labels) or otherlabel (e.g., biotin or an isotope). Polynucleotides provided or usedherein also include modified nucleic acids, particularly modified RNAs,which are disclosed in U.S. Pat. No. 9,464,124, which is incorporatedherein by reference in its entirety.

As used herein the term “synergistic” refers to an effect of combiningat least two factors that exceeds the sum of the effects obtained whenthe factors are not combined.

As used herein, the phrase “target plant gene” can refer to either agene located in the plant genome that is to be modified by gene editingmolecules provided in a system, method, composition and/or plant cellprovided herein or alternatively to a plant gene located in the plantgenome that is targeted for increased expression (e.g., an ODP2 and/oran WUS2 gene). Embodiments of target plant genes include(protein-)coding sequence, non-coding sequence, and combinations ofcoding and non-coding sequences. Modifications of a target plant geneinclude nucleotide substitutions, insertions, and/or deletions in one ormore elements of a plant gene that include a transcriptional enhancer orpromoter, a 5′ or 3′ untranslated region, a mature or precursor RNAcoding sequence, an intron, a splice donor and/or acceptor, a proteincoding sequence, a polyadenylation site, and/or a transcriptionalterminator. In certain embodiments, all copies or all alleles of a giventarget gene in a diploid or polyploid plant cell are modified to providehomozygosity of the modified target gene in the plant cell. Inembodiments, where a desired trait is conferred by a loss-of-functionmutation that is introduced into the target gene by gene editing, aplant cell, population of plant cells, plant, or seed is homozygous fora modified target gene with the loss-of-function mutation. In otherembodiments, only a subset of the copies or alleles of a given targetgene are modified to provide heterozygosity of the modified target genein the plant cell. In certain embodiments where a desired trait isconferred by a dominant mutation that is introduced into the target geneby gene editing, a plant cell, population of plant cells, plant, or seedis heterozygous for a modified target gene with the dominant mutation.Traits imparted by such modifications to certain plant target genesinclude improved yield, resistance to insects, fungi, bacterialpathogens, and/or nematodes, herbicide tolerance, abiotic stresstolerance (e.g., drought, cold, salt, and/or heat tolerance), proteinquantity and/or quality, starch quantity and/or quality, lipid quantityand/or quality, secondary metabolite quantity and/or quality, and thelike, all in comparison to a control plant that lacks the modification.The plant having a genome modified by gene editing molecules provided ina system, method, composition and/or plant cell provided herein differsfrom a plant having a genome modified by traditional breeding (i.e.,crossing of a male parent plant and a female parent plant), whereunwanted and random exchange of genomic regions as well as randommitotically or meiotically generated genetic and epigenetic changes inthe genome typically occurs during the cross and are then found in theprogeny plants. Thus, in embodiments of the plant (or plant cell) with amodified genome, the modified genome is more than 99.9% identical to theoriginal (unmodified) genome. In embodiments, the modified genome isdevoid of random mitotically or meiotically generated genetic orepigenetic changes relative to the original (unmodified) genome. Inembodiments, the modified genome includes a difference of epigeneticchanges in less than 0.01% of the genome relative to the original(unmodified) genome. In embodiments, the modified genome includes: (a) adifference of DNA methylation in less than 0.01% of the genome, relativeto the original (unmodified) genome; or (b) a difference of DNAmethylation in less than 0.005% of the genome, relative to the original(unmodified) genome; or (c) a difference of DNA methylation in less than0.001% of the genome, relative to the original (unmodified) genome. Inembodiments, the gene of interest is located on a chromosome in theplant cell, and the modified genome includes: (a) a difference of DNAmethylation in less than 0.01% of the portion of the genome that iscontained within the chromosome containing the gene of interest,relative to the original (unmodified) genome; or (b) a difference of DNAmethylation in less than 0.005% of the portion of the genome that iscontained within the chromosome containing the gene of interest,relative to the original (unmodified) genome; or (c) a difference of DNAmethylation in less than 0.001% of the portion of the genome that iscontained within the chromosome containing the gene of interest,relative to the original (unmodified) genome. In embodiments, themodified genome has not more unintended changes in comparison to theoriginal (unmodified) genome than 1×10{circumflex over ( )}-8 mutationsper base pair per replication. In certain embodiments, the modifiedgenome has not more unintended changes than would occur at the naturalmutation rate. Natural mutation rates can be determined empirically orare as described in the literature (Lynch, M., 2010; Clark et al.,2005).

To the extent to which any of the preceding definitions is inconsistentwith definitions provided in any patent or non-patent referenceincorporated herein by reference, any patent or non-patent referencecited herein, or in any patent or non-patent reference found elsewhere,it is understood that the preceding definition will be used herein.

Plant cells and related systems, methods, and compositions that providefor improved plant cell regenerative potential in comparison to controlplant cells are provided herein. In certain embodiments, improved plantcell regenerative potential is provided by transiently increasing theexpression of an ODP2 polypeptide and/or WUS2 polypeptide respectivelyencoded by an endogenous ODP2 gene or endogenous WUS2 gene in a plantgenome in the plant cells in comparison to a control plant cell. Suchtransient expression of the endogenous WUS2 and/or ODP2 genes canprovide for desired improvements in the regenerative capacity of theplant cell while avoiding undesired effects of increasing expression ofWUS2 and/or ODP2 in partially or fully regenerated plant structures,tissues, organs, and plants. Transient expression of the endogenous ODP2and/or WUS2 genes can be for a period of time and/or in an amountsufficient to result in improved regenerative potential in comparison toa control plant cell. In certain embodiments, the transient increase inthe expression of the endogenous ODP2 polypeptide and/or expression ofthe endogenous WUS2 polypeptide is for a period of about 1, 2, 4, 8, 12,16, 20, 24, 30, or 36 hours to about 72, 96, 120, 144, 168, 192, 276, or336 hours. In certain embodiments, the transient increase in theexpression of the endogenous ODP2 polypeptide and/or expression of theendogenous WUS2 polypeptide is for a period of about 2, 4, 8, 12, or 16hours to about 18, 20, 24, 30 or 36 hours. In certain embodiments, thetransient increase in the expression of the endogenous ODP2 polypeptideand/or expression of the endogenous WUS2 polypeptide is for a period ofabout 18, 20, 24, 30 or 36 hours to about 60, 80, 100, 120, 168, or 192hours. Such transient increases in expression of the endogenous ODP2and/or WUS2 genes can be measured by methods whereby accumulated ODP2and/or WUS2 gene products including mRNAs and/or proteins are measured.Useful methods of measuring ODP2 and/or WUS2 mRNAs include quantitativereverse transcriptase Polymerase Chain Reaction (qRT-PCR)-based and/orany hybridization-based assay. Useful methods for quantitating ODP2and/or WUS2 include immunoassays (e.g., ELISAs, RIAs) and/or massspectrometry-based methods. In certain embodiments, expression ofendogenous ODP2 and/or WUS2 gene products including mRNAs and/orproteins are transiently increased by at least 1.5-, 2-, 3-, 5-,10-fold, 20-fold, 50-fold, 100-fold, 500-fold, or 1000-fold incomparison to the corresponding endogenous ODP2 and/or WUS2 geneproducts in a control plant cell. In certain embodiments, expression ofendogenous ODP2 and/or WUS2 gene products including mRNAs and/orproteins are transiently increased by at least 1.5-, 2-, or 3-fold toabout 4-, 5-, 10-, 15-, 20-, 50-, 100-, 500-f, or 1000-fold incomparison to the corresponding endogenous ODP2 and/or WUS2 geneproducts in a control plant cell.

Endogenous ODP2 genes in plants that can be targeted for increasedexpression by methods provided herein include the endogenous ODP2 genesof both monocot and dicot plants. Such endogenous ODP2 genes include theODP2 genes that encode ODP2 peptides disclosed in US Patent ApplicationPublication Nos. 20190017061 and 20170121722, which are specificallyincorporated herein by reference in their entireties with respect tosuch disclosure of such ODP2 genes and peptides. Endogenous ODP2 genestargeted for increased expression can encode ODP2 peptides that compriseAPETALA2 (AP2) DNA binding motifs, and amino acid variants thereof. Incertain embodiments, the plant cell is a maize plant cell and theendogenous ODP2 gene targeted for increased expression encodes a ODP2polypeptide comprising an amino acid sequence having at least 95%, 96%,97%, or 99% amino acid sequence identity across the entire length of SEQID NO:1. In certain embodiments, the plant cell is a maize plant celland the endogenous ODP2 gene targeted for increased expression of theODP2 polypeptide is the endogenous maize ODP2 gene located on maizechromosome 3. In certain embodiments, the plant cell is a maize plantcell and the endogenous ODP2 gene targeted for increased expressioncomprises an endogenous polynucleotide that is operably linked to anendogenous maize ODP2 promoter of SEQ ID NO:3, SEQ ID NO:71, or anallelic variant thereof.

Endogenous WUS2 genes in plants that can be targeted by methods providedherein include the endogenous WUS2 genes of both monocot and dicotplants. Such endogenous WUS2 genes include the WUS2 genes that encodeWUS2 peptides disclosed in U.S. Pat. No. 7,256,322 and US PatentApplication Publication No. 20170121722, which are specificallyincorporated herein by reference in their entireties with respect tosuch disclosure of such WUS2 genes and peptides. Endogenous WUS2 genestargeted for increased expression can encode WUS2 peptides that compriseconserved homeodomain motifs such as the (E/R)TLPLFP motif (SEQ IDNO:109), the A(A/S)LEL(S/T)L motif (SEQ ID NO:110), a 25 amino acidmotif located between the (E/R)TLPLFP (SEQ ID NO:109) and theA(A/S)LEL(S/T)L (SEQ ID NO:110) motifs, and amino acid variants thereofIn certain embodiments, the plant cell is a maize plant cell and theendogenous WUS2 gene targeted for increased expression encodes a WUS2polypeptide comprising an amino acid sequence having at least 95%, 96%,97%, or 99% amino acid sequence identity across the entire length of SEQID NO:2. In certain embodiments, the plant cell is a maize plant celland the endogenous WUS2 gene targeted for increased expression of theendogenous WUS2 polypeptide the endogenous maize WUS2 gene located onmaize chromosome 10. In certain embodiments, the plant cell is a maizeplant cell and the endogenous WUS2 gene targeted for increasedexpression comprises an endogenous polynucleotide that is operablylinked to an endogenous maize WUS2 promoter of SEQ ID NO:4 or an allelicvariant thereof.

In certain embodiments, expression of the endogenous ODP2 and/or WUS2gene is transiently increased by introducing at least one exogenous genetranscription agent that stimulates transcription of the endogenous ODP2gene and/or with at least one exogenous gene transcription agent thatstimulates transcription of the endogenous WUS2 gene. In certainembodiments, expression of the endogenous ODP2 and/or WUS2 gene istransiently increased by introducing an exogenous gene transcriptionagent that stimulates transcription of both the endogenous ODP2 gene andthe endogenous WUS2 gene. In certain embodiments, additional exogenouspolynucleotides encoding an ODP2 and/or WUS2 polypeptide are notprovided to the cell since the exogenous transcription agents canincrease the regenerative capacity of the plant cell by increasingexpression of the endogenous ODP2 and/or WUS2 polypeptides encoded bythe endogenous ODP2 and/or WUS2 genes. Features of the exogenous genetranscription agents that can increase expression of the endogenous ODP2and WUS2 genes include: (a) a DNA binding domain that specifically bindsa sequence within the promoter or 5′ untranslated region (5′ UTR) of theendogenous ODP2 and/or WUS2 gene; (b) a transcriptional activationdomain (TAD) that is operably linked or operably associated with the DNAbinding domain; and, where required, (c) a nuclear localization signal(NLS) that is operably linked to the DNA binding domain. In certainembodiments, the aforementioned exogenous transcription factors areartificial transcription factors (ATFs). In certain embodiments, anexogenous gene transcription agent that stimulates transcription of boththe endogenous ODP2 gene and the endogenous WUS2 gene could comprise anartificial transcription factor comprising: (a) a first DNA bindingdomain that specifically binds the endogenous ODP2 gene promoter or 5′UTR, a second DNA binding domain that binds the endogenous WUS2 genepromoter or 5′ UTR; (b) a TAD that is operably linked or operablyassociated with the DNA binding domains, and, where required (c) an NLSthat is operably linked with the DNA binding domains. In certainembodiments, the ATFs comprise one or more of the features or elementswithin the features are wholly synthetic (e.g., non-naturally occurring)or wherein features from heterologous proteins are combined. Specificbinding to the promoter or 5′ untranslated region (5′ UTR) of theendogenous ODP2 and/or WUS2 gene by the DNA binding domain can be shownby DNA binding assays. Protein-DNA binding assays that can be usedinclude DNA electrophoretic mobility shift assays (EMSA); chromatinimmunoprecipitation (ChIP)-based assays; enzyme-linked immunoassays,fluorescence-anisotropy-based assays, and surface plasmon resonanceassays (Jantz and Berg, 2010). Specific DNA binding activity can also bedemonstrated in competitive DNA binding assays wherein binding to thetarget DNA sequence is inhibited more efficiently (e.g., at lowerconcentrations) by the target DNA sequence located within the promoteror 5′ untranslated region (5′ UTR) of the endogenous ODP2 or WUS2 genethan by an unrelated, non-target DNA sequence. In certain embodiments,the DNA binding domains and/or artificial transcription factors usedherein will bind the target DNA sequence with an affinity (K_(d)) of 10nM or less, 5 nM or less, 2 nM or less, or 1 nM or less or will bindwith an affinity of about 10 nM or 8 nM to about 1 nM or 0.5 nM. Otheroptional features of the artificial transcription factors includeepitope tags that can facilitate detection and/or quantitation ofexpression as well as cell penetrating peptides that can facilitateentry into a target plant cell.

Transcriptional activation domains (TADs) used in the ATFs can beobtained from either naturally occurring transcription factors or can bewholly or partially synthetic. Any of an acidic, glutamine-rich,proline-rich, isoleucine-rich, and/or an alanine-rich TAD can be used(Ma, 2011). Examples of such TADs that can be used include the maize C1,the VP16, and the VP64 transcription activation domains. In certainembodiments, multiple VP64 TADs can be used (Li et al., 2018). Anotherexample of a potent plant TAD that can be used in the ATFs providedherein is the EDLL motif that is found in AP2/ERF transcription factors(Tiwari et al., 2012). Yet another example of a potent plant TAD thatcan be used in the ATFs provided herein is a hybrid VP64-p65-Rtatripartite activator (VPR; SEQ ID NO: 91; Chavez et al., 2015).

Nuclear localization signals (NLS) that can be used in the ATF providedherein include monopartite and bipartite nuclear localization signals(Kosugi et al., 2009). Examples of monopartite NLS that can be usedinclude NLS that comprise at least 4 consecutive basic amino acids suchas the SV40 large T antigen NLS (PKKKRKV; SEQ ID NO:49) and anotherclass having only three basic amino acids with a K(K/R)X(K/R) consensussequence (SEQ ID NO:50). Examples of bipartite NLS that can be used inthe ATFs provided herein include (K/R)(K/R)X₁₀₋₁₂(K/R)_(3/5) (SEQ IDNO:51) where (K/R)_(3/5) represents at least three of either lysine orarginine of five consecutive amino acids. An NLS can also comprise aplant-specific class 5 NLS having a consensus sequence ofLGKR(K/R)(W/F/Y) (SEQ ID NO:52). Examples of specific NLS that can beused further include the maize opaque-2 nuclear localization signal andan extended SV40 large T antigen NLS (SEQ ID NO: 92).

In certain embodiments, the TAD and NLS elements can be operably linkedto the DNA binding domain in an ATF via either a direct covalent linkageof the elements and domain or by a use of a linker peptide or flexiblehinge polypeptide. Flexible hinge polypeptides include glycine-rich orglycine/serine containing peptide sequence. Such sequences can include,but are not limited to, a (Gly4)n sequence, a (Gly4Ser)n sequence of SEQID NO:53, a Ser(Gly4Ser)n sequence of SEQ ID NO:54, combinationsthereof, and variants thereof, wherein n is a positive integer equal to1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In certain embodiments, suchglycine-rich or glycine/serine containing hinge peptides can alsocontain threonyl and/or alanyl residues for flexibility as well as polarlysyl and/or glutamyl residues. Other examples of hinge peptides thatcan be used include immunoglobulin hinge peptides (Vidarsson et al.,2014).

A variety of cell-penetrating peptides (CPP) can also be used in the ATFprovided herein. CPPs that can be used include a minimal undecapeptideprotein transduction domain (corresponding to residues 47-57 of HIV-1TAT comprising YGRKKRRQRRR; SEQ ID NO:55); a polyarginine sequencecomprising a number of arginines sufficient to direct entry into a cell(e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain(Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); an DrosophilaAntennapedia protein transduction domain (Noguchi et al. (2003) Diabetes52(7): 1732-1737); a truncated human calcitonin peptide (Trehin et al.(2004) Pharm. Research 21: 1248-1256); polylysine (Wender et al. (2000)Proc. Natl. Acad. Sci. USA 97: 13003-13008); RRQRRTSKLMKR (SEQ IDNO:56); Transportan (e.g., GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:57));KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:58); and RQIKIWFQNRRMKWKK(SEQ ID NO:59). Exemplary CPP amino acid sequences also include

(SEQ ID NO: 60) YGRKKRRQRRR; (SEQ ID NO: 61)  RKKRRQRR; (SEQ ID NO: 62)YARAAARQARA; (SEQ ID NO: 63) THRLPRRRRRR; and (SEQ ID NO: 64)GGRRARRRRRR.

In certain embodiments, a TAD can be operably associated with a DNAbinding domain via a non-covalent interaction between a proteincomprising the TAD and the DNA binding peptide. In certain embodiments,such operable associations can be provided by protein domains that bindto one another (e.g., dimerization or other multimerization domains).Examples of such dimerization domains include leucine zipper structures.Such operable associates are similar to those used in yeast two-hybridsystems where interacting proteins are identified via their ability tojoin a TAD to a DNA binding domain (Bruckner et al., 2009). In certainembodiments, operable association can be achieved by using proteindomains that interact through binding a common ligand (e.g., theiDimerize™ Regulated Transcription System that uses Dmr A, B, or Cdimerization domains and ligands; Takara Bio, USA, Inc.). Suchligand-based systems have the advantage of allowing control ofdimerization (and activation of the endogenous ODP2 and/or WUS2 geneexpression) by ligand addition or removal.

In certain embodiments, the DNA binding domain can comprise anartificial zinc finger (AZF) DNA binding domain polypeptide whichspecifically binds a sequence within the promoter or 5′ untranslatedregion (5′ UTR) of the endogenous ODP2 and/or WUS2 gene. In certainembodiments, the AZF DNA binding domain specifically binds a target DNAsequence located within the promoter or 5′ untranslated region (5′ UTR)of the endogenous maize ODP2 and/or maize WUS2 gene. Such targetsequences in the endogenous maize ODP2 promoter include SEQ ID NO:6, SEQID NO:9, and any allelic variants thereof having one or more nucleotideinsertions, deletions, and/or substitutions found in other wild-typemaize genomes. AZF DNA binding domains predicted to bind the maize ODP2promoter target sequences of SEQ ID NO:6 or SEQ ID NO:9 include thepolypeptides comprising SEQ ID NO:5 and SEQ ID NO:8, respectively, aswell as variants thereof having at least 85%, 90%, 95%, 97%, 98%, or 99%sequence identity across the entire length of SEQ ID NO: 5 or 8; or oneor more conservative and/or semi-conservative amino acid substitutionsin SEQ ID NO: 5 or 8. Such target sequences in the endogenous maize WUS2promoter include SEQ ID NO:12, SEQ ID NO:15, DNA sequences in theendogenous maize WUS2 promoter corresponding to residues 100 to 225 or130 to 210 of SEQ ID NO:4 (or their complementary strand), SEQ IDNO:101, SEQ ID NO: 102 (in the minus or complementary strand of thedsDNA comprising SEQ ID NO: 4), and any allelic variants thereof havingone or more nucleotide insertions, deletions, and/or substitutions foundin other wild-type maize genomes. Such target sequences in theendogenous maize WUS2 promoter also include DNA sequences in theendogenous maize WUS2 promoter corresponding to residues 100 to 225 or130 to 210 of SEQ ID NO:4 (or their complementary strand), and anyallelic variants thereof having one or more nucleotide insertions,deletions, and/or substitutions found in other wild-type maize genomes.In certain embodiments, such allelic variants of the endogenous promoterWUS2 can have at least 80%, 85%, 90%, 95%, 97%, 98%, 98%, or 99%sequence identity to SEQ ID NO: 4, residues 100 to 225 or 130 to 210 ofSEQ ID NO:4, SEQ ID NO:101, or SEQ ID NO: 102. In certain embodiments,the target sequences in the endogenous maize WUS2 promoter also includeDNA sequences in the endogenous maize WUS2 promoter corresponding to:(i) residues 100, 105, 110, 115, 120, 125, 130, 132, 134, 135, 136, 137,or 138 to 155, 156, 157, 158, 160, 162, 165, or 170 of SEQ ID NO:4 (ortheir complementary strand); (ii) residues 171, 175, 180, 182, 183, 184,185, 186, 187, or 188 to 202, 203, 204, 205, 206, 207, 208, 209, 210,215, 220, or 225 of SEQ ID NO:4 (or their complementary strand); (iii)any combination of (i) and (ii); e.g., where two ATFs are used; or (iv)residues 100, 105, 110, 115, 120, 125, 130, 132, 134, 135, 136, 137, or138 to 202, 203, 204, 205, 206, 207, 208, 209, 210, 215, 220, or 225 ofSEQ ID NO:4 (or their complementary strand) of SEQ ID NO:4 (or theircomplementary strand). AZF-binding domains predicted to bind the maizeWUS2 promoter target sequences of SEQ ID NO:12 or SEQ ID NO:15 includethe polypeptides comprising SEQ ID NO:11 and SEQ ID NO:14, respectively,as well as variants thereof having at least 85%, 90%, 95%, 97%, 98%, or99% sequence identity across the entire length of SEQ ID NO:11 or 14; orone or more conservative and/or semi-conservative amino acidsubstitutions in SEQ ID NO:11 or 14. AZF DNA binding domains predictedto bind the maize WUS2 promoter target sequences comprising SEQ IDNO:101 or 102 include the polypeptides comprising SEQ ID NO: 105 or 106,respectively, as well as variants thereof having at least 85%, 90%, 95%,97%, 98%, or 99% sequence identity across the entire length of SEQ IDNO:105 or 106; or one or more conservative and/or semi-conservativeamino acid substitutions in SEQ ID NO:105 or 106. AZF DNA bindingdomains predicted to bind the maize WUS2 promoter target sequencescomprising SEQ ID NO:101, 102 and adjacent sequences or comprisingsubfragments (e.g., 9, 12, or 15 nucleotides) of SEQ ID NO:101 or 102and adjacent sequences also include variants of SEQ ID NO:105 or 106that further comprise additional zinc finger DNA binding motifs designedto bind the adjacent WUS2 promoter sequences. Artificial transcriptionfactors (ATFs) comprising the aforementioned AZF DNA-bindingpolypeptides can further comprise operably linked nuclear localizationpeptides, cell-penetrating peptides, and transcription activationdomains. Such ATFs predicted to bind and activate the endogenous ZmODP2promoter include the ATFs set forth in SEQ ID NO:7 and 10, as well asvariants thereof having at least 85%, 90%, 95%, 97%, 98%, or 99%sequence identity across the entire length of SEQ ID NO:7 or 10; orhaving at one or more conservative and/or semi-conservative amino acidsubstitutions in SEQ ID NO:7 or 10. Such ATFs predicted to bind andactivate the endogenous maize WUS2 (ZmWUS2) promoter include the ATFsset forth in SEQ ID NO:13 and 16, as well as variants thereof having atleast 85%, 90%, 95%, 97%, 98%, or 99% sequence identity across theentire length of SEQ ID NO:13 or 16; or having at one or moreconservative and/or semi-conservative amino acid substitutions in SEQ IDNO:13 or 16. Such ATFs predicted to bind and activate the endogenousmaize WUS2 (ZmWUS2) promoter include the ATFs set forth in SEQ ID NO:93and 95, as well as variants thereof having at least 85%, 90%, 95%, 97%,98%, or 99% sequence identity across the entire length of SEQ ID NO:93and 95; or having at one or more conservative and/or semi-conservativeamino acid substitutions in SEQ ID NO:93 and 95. In other embodiments,target AZF DNA binding sites in the promoter or 5′UTR sequences of otherendogenous plant ODP2 or WUS2 genes can be selected and AZF DNA bindingdomains as well as AZF transcription factors which specifically bind thetarget binding sites can be designed to obtain AZF transcription factorsthat can increase expression of other endogenous plant ODP2 or WUS2genes. In certain embodiments, target AZF DNA binding sites can beselected based on the presence of consecutive DNA triplets that can eachbe recognized by zinc finger domains comprising a Cysz-Hist zinc fingermotif. Target AZF DNA binding sites can be selected for the absence ofoverlap with sequences in non-target genes (e.g., genes other thanendogenous plant ODP2 or WUS2 genes). AZF DNA binding domains, includingvariants of the SEQ ID NO:11, 14, 105, and 106 AZF DNA binding domains,that target the selected AZF DNA binding sites can be constructed byjoining zinc finger domains. In certain embodiments, the AZF willcomprise about six (6) zinc finger domains joined by canonical TGEKP(SEQ ID NO: 48) linker peptides. In certain embodiments, rules governingthe design of Zn-ATFs to bind specific DNA sequences that have beenpublished (Sera and Uranga 2002; Gersbach, Gaj, and Barbas 2014) orprovided online (on the world wide web at “zincfingers.org/default2.htm”and “scripps.edu/barbas/zfdesign/zfdesignhome.php” can be applied to thedesign of the AZF's which bind target AZF binding sites in the ODP2 orWUS2 promoters or 5′UTR or to the construction of variants of the SEQ IDNO:11, 14, 105, and 106 AZF DNA binding domains. Features of artificialtranscription factors that comprise AZF-DNA binding domains for use inactivating endogenous genes in plants and other organisms that have beendescribed in various publications (van Tol and van der Zaal 2014;Heiderscheit et al. 2018; Van Eenennaam et al. 2004; Gupta et al. 2012;Stege et al. 2002; Sanchez et al. 2006; Holmes-Davis et al. 2005;Petolino and Davies 2013) can also be used in the design of artificialtranscription factors comprising AZF-DNA binding domains that recognizeplant ODP2 or WUS2 promoters or 5′UTR sequences or in the constructionof variants of the SEQ ID NO:11, 14, 105, and 106 AZF DNA bindingdomains.

In certain embodiments, the DNA binding domain can comprise anartificial transcription activator-like effector (TALE) DNA bindingpolypeptide (aTALE) which comprises an ‘repeat-variable di-residue’(RVD) containing domain that specifically binds a sequence within thepromoter or 5′ untranslated region (5′ UTR) of the endogenous ODP2 orWUS2 gene. In certain embodiments, the TALE DNA binding polypeptidespecifically binds a target DNA sequence located within the promoter or5′ untranslated region (5′ UTR) of the endogenous maize ODP2 or maizeWUS2 gene. Such target sequences in the endogenous maize ODP2 promoterinclude sequences within SEQ ID NO:3 or in SEQ ID NO:71, and any allelicvariants thereof having one or more nucleotide insertions, deletions,and/or substitutions found in other wild-type maize genomes. TALEDNA-binding polypeptides predicted to bind the maize ODP2 (ZmODP2)promoter target sequences within SEQ ID NO:3 include the polypeptidescomprising SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:81, SEQID NO:84, and SEQ ID NO:87, as well as variants thereof having at least85%, 90%, 95%, 97%, 98%, or 99% sequence identity across the entirelength of SEQ ID NO:23, 25, 27, 81, 84, or 87; or one or moreconservative and/or semi-conservative amino acid substitutions in SEQ IDNO: 23, 25, 27, 81, 84, or 87. Artificial transcription factors (ATFs)comprising the aforementioned TALE DNA binding polypeptides can furthercomprise operably linked nuclear localization peptides, cell-penetratingpeptides, and operably linked or operably associated transcriptionactivation domains. Such ATFs predicted to bind and activate theendogenous ZmODP2 promoter include the ATFs set forth in SEQ ID NO:24,26, 28, 82, 85, or 88, as well as variants thereof having at least 85%,90%, 95%, 97%, 98%, or 99% sequence identity across the entire length ofSEQ ID NO:24, 26, 28, 82, 85, or 88; or having at one or moreconservative and/or semi-conservative amino acid substitutions in SEQ IDNO:24, 26, 28, 82, 85, or 88. Any of the aforementioned ATFs predictedto bind the maize ODP2 promoter can be used either independently, intandem pairs of ATFs predicted to bind at ˜100 bp intervals in ZmODP2promoter sequences located 5′ to the ZmODP2 transcription start site, oras a set of three ATFs predicted to bind at ˜100 bp intervals in ZmODP2promoter sequences located 5′ to the ZmODP2 transcription start site. Inother embodiments, ATFs predicted to bind the maize ODP2 promoter can beused in tandem pairs of ATFs predicted to bind at ˜50 bp intervals inZmODP2 promoter sequences located 5′ to the ZmODP2 transcription startsite, or as a set of three ATFs predicted to bind at ˜50 bp intervals inZmODP2 promoter sequences located 5′ to the ZmODP2 transcription startsite. Such target sequences in the endogenous maize WUS2 promoterinclude sequences located within SEQ ID NO:4 and any allelic variantsthereof having one or more nucleotide insertions, deletions, and/orsubstitutions found in other wild-type maize genomes. TALE DNA-bindingpolypeptides predicted to bind the maize WUS2 promoter target sequenceswithin SEQ ID NO:4 include the polypeptides comprising SEQ ID NO:17, SEQID NO:19, SEQ ID NO:21, SEQ ID NO:72, SEQ ID NO:75, and SEQ ID NO:78, aswell as variants thereof having at least 85%, 90%, 95%, 97%, 98%, or 99%sequence identity across the entire length of SEQ ID NO:17, 19, 21, 72,75, or 78; or one or more conservative and/or semi-conservative aminoacid substitutions in SEQ ID NO: 17, 19, 21, 72, 75, or 78. Artificialtranscription factors (ATFs) comprising the aforementioned TALEDNA-binding polypeptides can further comprise operably linked nuclearlocalization peptides and transcription activation domains. Such ATFspredicted to bind and activate the endogenous ZmWUS2 promoter includethe ATFs set forth in SEQ ID NO:18, 20, 22, 73, 76, or 79, as well asvariants thereof having at least 85%, 90%, 95%, 97%, 98%, or 99%sequence identity across the entire length of SEQ ID NO:18, 20, 22, 73,76, or 79; or having at one or more conservative and/orsemi-conservative amino acid substitutions in SEQ ID NO:18, 20, 22, 73,76, or 79. Any of the aforementioned ATFs comprising the aforementionedTALE DNA-binding polypeptides predicted to bind the maize WUS2 (ZmWUS2)promoter can be used either independently, in tandem pairs of ATFspredicted to bind at ˜100 bp intervals in the maize WUS2 promotersequences located 5′ to the ZmWUS2 transcription start site, or as a setof three ATFs predicted to bind at ˜100 bp intervals in ZmWUS2 promotersequences located 5′ to the ZmWUS2 transcription start site. In otherembodiments, ATFs predicted to bind the maize WUS2 promoter can be usedin tandem pairs of ATFs predicted to bind at ˜50 bp intervals in ZmWUS2promoter sequences located 5′ to the ZmWUS2 transcription start site, oras a set of three ATFs predicted to bind at ˜50 bp intervals in ZmWUS2promoter sequences located 5′ to the ZmWUS2 transcription start site. Inother embodiments, target TALE DNA binding sites in the promoter or5′UTR sequences of other endogenous plant ODP2 or WUS2 genes can beselected and TALE DNA binding domains as well as TALE transcriptionfactors which specifically bind the target binding sites can be designedto obtain TALE transcription factors that can increase expression ofother endogenous plant ODP2 or WUS2 genes. In certain embodiments, rulesgoverning the design of TALEs to bind specific DNA sequences that havebeen published (Moore, Chandrahas, and Bleris 2014; Čermák et al. 2017;Sanjana et al. 2012; Thakore and Gersbach 2016) or provided online (onthe https internet site “tale-nt.cac.cornell.edu/node/add/single-tale”)can be applied to the design of the TALE's which bind target TALEbinding sites in the ODP2 or WUS2 promoters or 5′UTR.

In certain embodiments, the DNA binding domain can comprise a complex ofan RNA guided DNA binding polypeptide that is nuclease activitydeficient and a guide RNA comprising a tracrRNA and crRNA polynucleotidesequence which corresponds to a sequence immediately adjacent to the 5′end of a protospacer adjacent motif (PAM) in the target ODP2 or WUS2promoter or 5′ UTR, where the complex specifically binds a sequencewithin the promoter or 5′ untranslated region (5′ UTR) of the endogenousODP2 or WUS2 gene. RNA guided DNA binding polypeptides that are nucleaseactivity deficient are also referred to herein as nuclease activitydeficient RNA-guided DNA binding polypeptides (ndRGDBP). In certainembodiments, the guide RNA is a single guide RNA (sgRNA) where the crRNAand the tracrRNA are covalently linked. In other embodiments, a dualguide RNA can be used where the crRNA and the tracrRNA are notcovalently linked. In general, the crRNA typically comprises about an 18or 19 to about a 21 or 22 nucleotide sequence which corresponds to thesequence immediately adjacent to the 5′ end of a protospacer adjacentmotif (PAM) (e.g., for Cas9 and similar RNA directed nucleases). Ingeneral, the crRNA typically comprises about a 20, 21, 22, 23, or 24nucleotide sequence which corresponds to the sequence immediatelyadjacent to the 3′ end of a PAM (e.g., for Cas12a (i.e., Cpf1) andsimilar RNA directed nucleases). Nuclease activity deficient RNA guidedDNA binding polypeptides (ndRGDBP) used in such complexes can compriseRNA guided nucleases (Cas or Cas12a nucleases) having mutations thatrender the protein nuclease activity deficient (e.g., having a 99% orgreater reduction in nuclease activity under physiological conditions ina plant cell nucleus). Such nuclease deficient variants of Cas like Cas9or Cas12a proteins are referred to herein and elsewhere as “dCas” (e.g.,dCas9, dCasJ, and the like) or “dCpf1” or “dCas12a” proteins (i.e.,“dead Cas” or “dead Cpf1” or “dead Cas12a”). Domains in Cas or Cas12aproteins which can be disrupted to reduce or eliminate nuclease activityinclude HNH and RuvC-like nuclease domains. Mutations in the catalyticresidues of the HNH and RuvC-like nuclease domains of Cas proteins canprovide for nuclease-deficient RNA-guided DNA binding proteins (Jinek etal., 2012; Schindele et al., 2018). Examples of such mutations includethe D10A and H840A mutations in the Cas9 protein and analogous mutationsin the corresponding residues of other Cas9-like proteins identified byalignment with the Cas9 protein. Examples of a dCas9 protein include thepolypeptide of SEQ ID NO:29. Other dCas proteins can be obtained byinactivation of nuclease domains include the dCasJ mutants obtained bymutating the CasJ protein of SEQ ID NO:47. Mutations in the nucleasedomain of the CasJ include D901A and/or E1228A amino acid substitutionsin the CasJ protein of SEQ ID NO:47 and analogous mutations in thecorresponding residues of other CasJ proteins identified by alignmentwith the CasJ protein of SEQ ID NO:47. Mutations in the RuvC-likenuclease domains of Cas12a (i.e., Cpfl) proteins can provide for dCas12anuclease-deficient RNA-guided DNA binding proteins. Examples of suchmutations include the E993A mutation in the AsCpfl protein (Zhang et al,2017), the D917A, E1006A, E1028A, D1255A, and/or N1257A mutations in theAsCpf1 protein of SEQ ID NO:44, the D832A, E925A, and/or D1148Amutations in the LbCpfl protein of SEQ ID NO:45, the D917A, E1006A,E1028A, D1255A, and/or N1257A mutations in the FnCpf1 protein of SEQ IDNO:46, and analogous mutations in the corresponding residues of otherCpf1 proteins identified by alignment with the Cpf1 proteins of SEQ IDNO:44, 45, or 46. Any of the aforementioned dCas9, dCas, dCasJ, or dCpf1proteins can be used in artificial transcription factors (ATFs) providedherein that further comprise transcription activation domains,cell-penetrating peptides, and nuclear localization domains. Examples ofsuch ATFs include the dCas9 ATF set forth in SEQ ID NO:30, SEQ ID NO:90,and variants thereof that retain RNA guided DNA binding activity andthat are nuclease activity deficient. Such dCas9 ATF variants thatretain RNA guided DNA binding activity and that are nuclease activitydeficient include variants thereof having at least 85%, 90%, 95%, 97%,98%, or 99% sequence identity across the entire length of SEQ ID NO:30or 90; or having at one or more conservative and/or semi-conservativeamino acid substitutions in SEQ ID NO:90. Such artificial transcriptionfactors are used with guide RNAs (sgRNAs or crRNAs and a tracrRNA) toform an ATF/guide RNA complex which can specifically bind sequences inthe plant ODP2 and/or WUS2 promoters or 5′ UTR that is immediatelyadjacent to a protospacer adjacent motif (PAM) sequence. Guide RNAs thatdirect the dCas or dCpf1 ATF proteins to endogenous plant ODP2 or WUS2genes can be obtained by identifying target sequences adjacent to PAMsequences in the plant ODP2 and/or WUS2 promoters or 5′ UTR andsynthesizing a crRNA or sgRNA that is complementary to that sequence.The type of RNA-guided DNA binding program typically informs thelocation of suitable PAM sites and design of crRNAs or sgRNAs. G-richPAM sites, e.g., 5′-NGG are typically targeted for design of crRNAs orsgRNAs used with dCas9 proteins. T-rich PAM sites (e.g., 5′-TTTV [1],where “V” is A, C, or G) are typically targeted for design of crRNAs orsgRNAs used with dCpf1 proteins. PAM sites including TTN, CTN, TCN, CCN,TTTN, TCTN, TTCN, CTTN, ATTN, TCCN, TTGN, GTTN, CCCN, CCTN, TTAN, TCGN,CTCN, ACTN, GCTN, TCAN, GCCN, and CCGN targeted for design of crRNAs orsgRNAs used with dCasJ proteins (e.g., SEQ ID NO:47). Such crRNAs orsgRNAs can be complementary to sequences that are immediately adjacentto PAM sequences located on either strand of the OPD2 or WUS2 promoter.In certain embodiments the dCas or dCpf1 ATF proteins and guide RNAs areprovided to the cell as a pre-assembled ribonucleoprotein (RNP) complex.For example, the ATF can be expressed in an expression host (e.g., E.coli), purified, and complexed with the guide RNA. In other embodiments,the dCas or dCpf1 ATF proteins and guide RNAs are provided separately tothe plant cell. Guide RNAs that are synthesized and optionally includingchemically modified ribonucleotides can also be used (O'Reilly et al.,2018; Yin et al., 2018). In other embodiments, the dCas or dCpf1 ATFproteins and guide RNAs are provided by introducing one or morepolynucleotides encoding the dCas or dCpf1 ATF proteins and/or guideRNA(s) into the target plant cell. In certain embodiments, the guideRNAs are provided to the plant cell by introducing polynucleotidescomprising a class III RNA polymerase III promoter that is operablylinked to the DNA encoding the guide RNA (Long et al., 2018). Such RNApolymerase III promoters include U6 promoters from monocot plants (e.g.,OsU6a, OsU6b, and OsU6c from rice) or dicot plants (e.g., GmU6 fromsoybean, GhU6 from cotton, and AtU6-1 or AtU6-29 from Arabidopsisthaliana). Useful U6 promoters from maize, tomato, or soybean aredisclosed in WO 2015/131101, which is incorporated herein by referencein its entirety with respect to such promoters and their use. In certainembodiments, the plant cell is a maize plant and an ATF/guide RNAcomplex that specifically binds target DNA sequence located within thepromoter or 5′ untranslated region (5′ UTR) of the endogenous maize ODP2or maize WUS2 gene. Expression of the guide RNA can in certainembodiments be driven by a plant U6 spliceosomal RNA promoter, which canbe native to the genome of the plant cell or from a different species,and claiming priority to U.S. Provisional Patent Application 61/945,700,incorporated herein by reference, or a homologue thereof; such apromoter is operably linked to DNA encoding the guide RNA for directingan endonuclease, followed by a suitable 3′ element such as a U6 poly-Tterminator. In another embodiment, an expression cassette for expressingguide RNAs in plants is used, wherein the promoter is a plant U3, 7SL(signal recognition particle RNA), U2, or U5 promoter, or chimericsthereof, e.g., as described in WO 2015/131101), incorporated herein byreference. Guide RNAs that can be used to target a dCas9 ATF to anendogenous maize ODP2 promoter can comprise crRNA molecules encoded byDNA molecules set forth as SEQ ID NO:31, 32, 33, 34, or 35. In certainembodiments, the dCas9 ATF and only one guide RNA comprising a crRNAencoded by SEQ ID NO:31, 32, 33, 34, or 35 are introduced into the maizeplant cell to activate transcription of the endogenous maize ODP2 gene.In other embodiments, the dCas9 ATF and two, three, four, or five guideRNAs each comprising one crRNA molecules encoded by SEQ ID NO:31, 32,33, 34, and/or 35 are introduced into the maize plant cell to activatetranscription of the endogenous maize ODP2 gene. Guide RNAs that can beused to target a dCas9 ATF to an endogenous maize WUS2 promoter cancomprise crRNA molecules encoded by DNA molecules set forth as SEQ IDNO:36, 37, 38, 39, or 40. In certain embodiments, the dCas9 ATF and onlyone guide RNA comprising a crRNA encoded by SEQ ID NO: 36, 37, 38, 39,or 40 are introduced into the maize plant cell to activate transcriptionof the endogenous maize WUS2 gene. In other embodiments, the dCas9 ATFand two, three, four, or five guide RNAs each comprising one crRNAmolecule encoded by SEQ ID NO:31, 32, 33, 34, and/or 35 are introducedinto the maize plant cell to activate transcription of the endogenousmaize WUS2 gene. In certain embodiments, the crRNAs SEQ ID NO:31, 32,33, 34, 35, 36, 37, 38, 39, or 40 further comprise a covalently linkedtracrRNA and are thus provided as sgRNAs. In other embodiments, thecrRNAs SEQ ID NO:31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 are providedwith a non-covalently linked tracrRNA to provide a dual guide RNA. Ininstances where an allelic variant of an endogenous maize ODP2 or WUS2promoter that differs in sequence by one or more insertions, deletions,and/or substitutions from SEQ ID NO:3 or SEQ ID NO:4, respectively, acorresponding crRNA can be synthesized that is complementary to theallelic variant sequence and used in a single or dual guide RNA with adCas ATF to activate transcription of the endogenous maize ODP2 gene orWUS2 gene that comprises the allelic variant promoter sequence.

In certain embodiments, the expression of the endogenous ODP2 and/orWUS2 genes are increased in isolated plant cells or plant protoplasts(i.e., are not located in undissociated or intact plant tissues, plantparts, or whole plants). In certain embodiments, the plant cells areobtained from any plant part or tissue or callus. In certainembodiments, the culture includes plant cells obtained from a planttissue, a cultured plant tissue explant, whole plant, intact nodal bud,shoot apex or shoot apical meristem, root apex or root apical meristem,lateral meristem, intercalary meristem, seedling, whole seed, halvedseed or other seed fragment, zygotic embryo, somatic embryo, immatureembryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf,petiole, stem, tuber, root, callus, or plant cell suspension. In certainembodiments, the plant cell is derived from the L1 or L2 layer of animmature or mature embryo of a monocot plant (e.g., maize, wheat,sorghum, or rice).

In certain embodiments, the expression of the endogenous ODP2 and/orWUS2 genes are increased in plant cells that are located inundissociated or intact plant tissues, plant parts, plant explants, orwhole plants. In certain embodiments, the plant cell can be located inan intact nodal bud, a cultured plant tissue explant, shoot apex orshoot apical meristem, root apex or root apical meristem, lateralmeristem, intercalary meristem, seedling, whole seed, halved seed orother seed fragment, zygotic embryo, somatic embryo, immature embryo,ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole,stem, tuber, root, or callus. In certain embodiments, the explants usedinclude immature embryos. Immature embryos (e.g., immature maizeembryos) include, 1.8-2.2 mm embryos, 1-7 mm embryos, and 3-7 mmembryos,. In certain embodiments, the aforementioned embryos areobtained from mature ear-derived seed, leaf bases, leaves from matureplants, leaf tips, immature inflorescences, tassels, immature ears, andsilks. In various aspects, the plant-derived explant used fortransformation includes immature embryos, 1.8-2.2 mm embryos, 1-7 mmembryos, and 3.5-7 mm embryos. In an aspect, the embryos used in thedisclosed methods can be derived from mature ear-derived seed, leafbases, leaves from mature plants, leaf tips, immature inflorescences,tassel, immature ear, or silks. In certain embodiments, the plant cellis a pluripotent plant cell (e.g., a stem cell or meristem cell). Incertain embodiments, the plant cell is located within the L1 or L2 layerof an immature or mature embryo of a monocot plant (e.g., maize, wheat,sorghum, or rice). In certain embodiments, methods of editing genomes ofwhole plants, seeds, embryos, explants, or meristematic tissue publishedin WO2018085693, which is incorporated herein by reference in itsentirety, can be adapted for use in the plant cells and related systems,methods, compositions, or cultures provided herein.

In certain embodiments, the plant cells can comprise haploid, diploid,or polyploid plant cells or plant protoplasts, for example, thoseobtained from a haploid, diploid, or polyploid plant, plant part ortissue, or callus. In certain embodiments, plant cells in culture (orthe regenerated plant, progeny seed, and progeny plant) are haploid orcan be induced to become haploid; techniques for making and usinghaploid plants and plant cells are known in the art, see, e.g., methodsfor generating haploids in Arabidopsis thaliana by crossing of awild-type strain to a haploid-inducing strain that expresses alteredforms of the centromere-specific histone CENH3, as described byMaruthachalam and Chan in “How to make haploid Arabidopsis thaliana”,protocol available atwww[dot]openwetware[dot]org/images/d/d3/Haploid_Arabidopsis_protocol[dot]pdf;(Ravi et al. (2014) Nature Communications, 5:5334, doi:10.1038/ncomms6334). Haploids can also be obtained in a wide variety ofmonocot plants (e.g., maize, wheat, rice, sorghum, barley) or dicotplants (e.g., soybean, Brassica sp. including canola, cotton, tomato) bycrossing a plant comprising a mutated CENH3 gene with a wildtype diploidplant to generate haploid progeny as disclosed in U.S. Pat. No.9,215,849, which is incorporated herein by reference in its entirety.Haploid-inducing maize lines that can be used to obtain haploid maizeplants and/or cells include Stock 6, MHI (Moldovian Haploid Inducer),indeterminate gametophyte (ig) mutation, KEMS, RWK, ZEM, ZMS, KMS, andwell as transgenic haploid inducer lines disclosed in U.S. Pat. No.9,677,082, which is incorporated herein by reference in its entirety.Examples of haploid cells include but are not limited to plant cellsobtained from haploid plants and plant cells obtained from reproductivetissues, e.g., from flowers, developing flowers or flower buds, ovaries,ovules, megaspores, anthers, pollen, megagametophyte, and microspores.In certain embodiments where the plant cell or plant protoplast ishaploid, the genetic complement can be doubled by chromosome doubling(e.g., by spontaneous chromosomal doubling by meiotic non-reduction, orby using a chromosome doubling agent such as colchicine, oryzalin,trifluralin, pronamide, nitrous oxide gas, anti-microtubule herbicides,anti-microtubule agents, and mitotic inhibitors) in the plant cell orplant protoplast to produce a doubled haploid plant cell or plantprotoplast wherein the complement of genes or alleles is homozygous; yetother embodiments include regeneration of a doubled haploid plant fromthe doubled haploid plant cell or plant protoplast. Another embodimentis related to a hybrid plant having at least one parent plant that is adoubled haploid plant provided by this approach. Production of doubledhaploid plants provides homozygosity in one generation, instead ofrequiring several generations of self-crossing to obtain homozygousplants. The use of doubled haploids is advantageous in any situationwhere there is a desire to establish genetic purity (i.e. homozygosity)in the least possible time. Doubled haploid production can beparticularly advantageous in slow-growing plants, such as fruit andother trees, or for producing hybrid plants that are offspring of atleast one doubled-haploid plant.

In certain embodiments, the plant cells where expression of theendogenous ODP2 and/or WUS2 genes are increased, as well as the relatedmethods, systems, compositions, or reaction mixtures provided herein caninclude plant cells obtained from or located in any monocot or dicotplant species of interest, for example, row crop plants, fruit-producingplants and trees, vegetables, trees, and ornamental plants includingornamental flowers, shrubs, trees, groundcovers, and turf grasses. Incertain non-limiting embodiments, the plant cells are obtained from orlocated in alfalfa (Medicago sativa), almonds (Prunus dulcis), apples(Malus x domestica), apricots (Prunus armeniaca, P. brigantine, P.mandshurica, P. mume, P. sibirica), asparagus (Asparagus officinalis),bananas (Musa spp.), barley (Hordeum vulgare), beans (Phaseolus spp.),blueberries and cranberries (Vaccinium spp.), cacao (Theobroma cacao),canola and rapeseed or oilseed rape, (Brassica napus), carnation(Dianthus caryophyllus), carrots (Daucus carota sativus), cassava(Manihot esculentum), cherry (Prunus avium), chickpea (Cider arietinum),chicory (Cichorium intybus), chili peppers and other capsicum peppers(Capsicum annuum, C. frutescens, C. chinense, C. pubescens, C.baccatum), chrysanthemums (Chrysanthemum spp.), coconut (Cocosnucifera), coffee (Coffea spp. including Coffea arabica and Coffeacanephora), cotton (Gossypium hirsutum L.), cowpea (Vigna unguiculata),cucumber (Cucumis sativus), currants and gooseberries (Ribes spp.),eggplant or aubergine (Solanum melongena), eucalyptus (Eucalyptus spp.),flax (Linum usitatissumum L.), geraniums (Pelargonium spp.), grapefruit(Citrus x paradisi), grapes (Vitus spp.) including wine grapes (Vitusvinifera), guava (Psidium guajava), hemp and cannabis (e.g., Cannabissativa and Cannabis spp.), hops (Humulus lupulus), irises (Iris spp.),lemon (Citrus limon), lettuce (Lactuca sativa), limes (Citrus spp.),maize (Zea mays L.), mango (Mangifera indica), mangosteen (Garciniamangostana), melon (Cucumis melo), millets (Setaria spp, Echinochloaspp, Eleusine spp, Panicum spp., Pennisetum spp.), oats (Avena sativa),oil palm (Ellis quineensis), olive (Olea europaea), onion (Allium cepa),orange (Citrus sinensis), papaya (Carica papaya), peaches and nectarines(Prunus persica), pear (Pyrus spp.), pea (Pisa sativum), peanut (Arachishypogaea), peonies (Paeonia spp.), petunias (Petunia spp.), pineapple(Ananas comosus), plantains (Musa spp.), plum (Prunus domestica),poinsettia (Euphorbia pulcherrima), Polish canola (Brassica rapa),poplar (Populus spp.), potato (Solanum tuberosum), pumpkin (Cucurbitapepo), rice (Oryza sativa L.), roses (Rosa spp.), rubber (Heveabrasiliensis), rye (Secale cereale), safflower (Carthamus tinctorius L),sesame seed (Sesame indium), sorghum (Sorghum bicolor), soybean (Glycinemax L.), squash (Cucurbita pepo), strawberries (Fragaria spp., Fragariax ananassa), sugar beet (Beta vulgaris), sugarcanes (Saccharum spp.),sunflower (Helianthus annus), sweet potato (Ipomoea batatas), tangerine(Citrus tangerina), tea (Camellia sinensis), tobacco (Nicotiana tabacumL.), tomato (Lycopersicon esculentum), tulips (Tulipa spp.), turnip(Brassica rapa rapa), walnuts (Juglans spp. L.), watermelon (Citruluslanatus), wheat (Tritium aestivum), or yams (Discorea spp.).

In certain embodiments, the plant cells where the expression of theendogenous ODP2 and/or WUS2 genes are increased can be plant cells thatare (a) encapsulated or enclosed in or attached to a polymer (e.g.,pectin, agarose, or other polysaccharide) or other support (solid orsemi-solid surfaces or matrices, or particles or nanoparticles); (b)encapsulated or enclosed in or attached to a vesicle or liposome orother fluid compartment; or (c) not encapsulated or enclosed orattached. In certain embodiments, the plant cells can be in liquid orsuspension culture, or cultured in or on semi-solid or solid media, orin a combination of liquid and solid or semi-solid media (e.g., plantcells or protoplasts cultured on solid medium with a liquid mediumoverlay, or plant cells or protoplasts attached to solid beads or amatrix and grown with a liquid medium). In certain embodiments, theplant cells encapsulated in a polymer (e.g., pectin, agarose, or otherpolysaccharide) or other encapsulating material, enclosed in a vesicleor liposome, suspended in a mixed-phase medium (such as an emulsion orreverse emulsion), or embedded in or attached to a matrix or other solidsupport (e.g., beads or microbeads, membranes, or solid surfaces).

In a related aspect, the disclosure provides arrangements of plant cellshaving improved plant cell regenerative potential in the systems,methods, and compositions described herein, such as arrangements ofplant cells convenient for screening purposes or for high-throughputand/or multiplex transformation or gene editing experiments. In anembodiment, the disclosure provides an arrangement of multiple plantcells comprising: (a) an exogenous gene transcription agent whichtransiently increases expression of an endogenous ODP2 polypeptideand/or an exogenous gene transcription agent which increases expressionof an endogenous WUS2 polypeptide; and optionally (b) genome alteringreagent(s). In certain embodiments, the arrangements of plant cells canfurther comprise at least one chemical, enzymatic, or physical deliveryagent. In another embodiment, the disclosure provides an array includinga plurality of containers, each including at least one plant cell orplant protoplast having improved plant cell regenerative potential. Inan embodiment, the disclosure provides arrangements of plant cellshaving the exogenous gene transcription agent(s) and optionally thegenome altering reagents, wherein the plant cells are in an arrayedformat, for example, in multi-well plates, encapsulated or enclosed invesicles, liposomes, or droplets (useful, (e.g., in a microfluidicsdevice), or attached discretely to a matrix or to discrete particles orbeads; a specific embodiment is such an arrangement of multiple plantcells having improved plant cell regenerative potential provided in anarrayed format, further including at least one genome alteringreagent(s) (e.g., an RNA-guided DNA nuclease, at least one guide RNA, ora ribonucleoprotein including both an RNA-guided DNA nuclease and atleast one guide RNA), which may be different for at least some locationson the array or even for each location on the array, and optionally atleast one chemical, enzymatic, or physical delivery agent.

In the systems and methods provided herein, plant cells can be exposedto one exogenous gene transcription agent which transiently increasesexpression of an endogenous ODP2 polypeptide and/or at least oneexogenous gene transcription agent which increases expression of anendogenous WUS2 polypeptide and/or genome altering reagents in anytemporal order. In certain embodiments, the genome altering reagents andaforementioned exogenous gene transcription agent(s) are providedsimultaneously. In other embodiments, the genome altering reagents areprovided after the exogenous gene transcription agent(s) are provided.In other embodiments, the genome altering reagents are provided beforethe exogenous gene transcription agent(s) are provided. In summary, thegenome altering reagents can be provided to a plant cell either previousto, concurrently with, or subsequent to exposing the plant cell to theexogenous gene transcription agent(s).

Plant cells having improved plant cell regenerative potential conferredby an increase in the transient expression of the endogenous ODP2 and/orWUS2 genes are provided herein. Also provided by the disclosure arecompositions derived from or grown from the plant cell or plantprotoplast having improved plant cell regenerative potential, providedby the systems and methods disclosed herein; such compositions includemultiple protoplasts or cells, callus, a somatic embryo, a somaticmeristem, embryogenic callus, or a regenerated plant grown from theplant cell or plant protoplast having improved plant cell regenerativepotential. Improved plant cell regenerative potential in plant cellsthat have been subjected to a transient increase in ODP2 and/or WUS2gene expression can be assessed by a variety of techniques. In certainembodiments, such techniques can compare the numbers and/or amount ofregenerable plant structures (e.g., immature embryos, somatic embryos,embryogenic calli, somatic meristems, organogenic calli, shoots, orshoots further comprising roots) formed and/or recovered from a givennumber of plant cells subjected to the transient increase in endogenousODP2 and/or WUS2 gene expression versus control plant cells that werenot subjected to the transient increase in ODP2 and/or WUS2 geneexpression. In certain embodiments, it is understood that the plantcells can be directly subjected to the transient increase in endogenousODP2 and/or WUS2 gene expression (e.g., by or indirectly (e.g., byexposure, contact, or other signaling of neighboring cells The principleattributes of tissues targeted for transient expression of the ATFsprovided would be the presence of dividing cells and the ability to growin tissue culture media. These tissues include, but are not limited todividing cells from young maize leaf, meristems and scutellar tissuefrom about 8 or 10 to about 12 or 14 days after pollination (DAP)embryos. The isolation of maize embryos has been described in severalpublications (Brettschneider, Becker, and Lörz 1997; Leduc et al. 1996;Frame et al. 2011; K. Wang and Frame 2009). In certain embodiments,basal leaf tissues (e.g., leaf tissues located about 0 to 3 cm from theligule of a maize plant; Kirienko, Luo, and Sylvester 2012) are targetedfor transient expression of the ATFs. In certain embodiments, suchincreases in numbers and/or amounts of regenerable plant structures canbe observed in about 1, 2, or 3 to about 7, 10, 14, 30, or 60 daysfollowing the transient increase in endogenous ODP2 and/or WUS2 geneexpression. Methods for obtaining regenerable plant structures andregenerating plants from the plant cells provided herein can be adaptedfrom methods disclosed in US Patent Application Publication No.20170121722, which is incorporated herein by reference in its entiretyand specifically with respect to such disclosure. In certainembodiments, single plant cells subjected to the transient increase inendogenous ODP2 and/or WUS2 gene expression will give rise to singleregenerable plant structures. In certain embodiments, the singleregenerable plant cell structure can form from a single cell on, orwithin, an explant that has been subjected to the transient increase inendogenous ODP2 and/or WUS2 gene expression and optionally subjected totreatment with a genome altering reagent. In certain embodiments,initiation or formation of the single plant cell regenerable structurecan occur where single-cell-derived cell or tissue proliferation (e.g.,growth of callus, non-differentiated callus, embryogenic callus andorganogenic callus) occurring before initiation of the regenerable plantstructure is reduced or absent. In certain embodiments, regenerableplant structures from plant cells subjected to the transient increase inendogenous ODP2 and/or WUS2 gene expression and optionally a genomealtering reagent can be form the regenerable plant structure in theabsence of exogenous cytokinin or with levels of cytokinin that arelower than those required to initiate formation of the regenerablestructure from a control cell. In certain embodiments, regenerable plantstructures from plant cells subjected to the transient increase inendogenous ODP2 and/or WUS2 gene expression and optionally a genomealtering reagent can be identified and/or selected via a positive growthselection based on the ability of those plant cells to initiate and/orform the regenerable plant structures more rapidly than adjacent plantcells that have not been subjected to the transient increase inendogenous ODP2 and/or WUS2 gene expression. In certain embodiments,such positive growth selection can obviate or reduce the need to use atraditional negative selection system where an antibiotic or herbicideis used to inhibit growth of adjacent, non-transformed cells that do notcontain a gene that confers resistance to the antibiotic or herbicide.Nonetheless, embodiments where a selectable marker gene conferringresistance to an antibiotic, herbicide, or other agent can be introducedinto the plant cell at least temporarily during initiation and/orformation of the regenerable plant cell structures to facilitateidentification and recovery.

In some embodiments, methods provided herein can include the additionalstep of growing or regenerating a plant from a plant cell that had beensubjected to an increase in the transient expression of the endogenousODP2 and/or WUS2 genes or from a regenerable plant structure obtainedfrom that plant cell. In certain embodiments, the plant can furthercomprise an inserted transgene, a target gene edit, or genome edit asprovided by the methods and compositions disclosed herein. In certainembodiments, callus is produced from the plant cell, and plantlets andplants produced from such callus. In other embodiments, whole seedlingsor plants are grown directly from the plant cell without a callus stage.Thus, additional related aspects are directed to whole seedlings andplants grown or regenerated from the plant cell or plant protoplasthaving a target gene edit or genome edit, as well as the seeds of suchplants. In certain embodiments wherein the plant cell or plantprotoplast is subjected to genetic or epigenetic modification (forexample, stable or transient expression of a transgene, gene silencing,epigenetic silencing, or genome editing by means of, e.g., an RNA-guidedDNA nuclease), the grown or regenerated plant exhibits a phenotypeassociated with the genetic or epigenetic modification. In certainembodiments, the grown or regenerated plant includes in its genome twoor more genetic or epigenetic modifications that in combination provideat least one phenotype of interest. In certain embodiments, aheterogeneous population of plant cells having a target gene edit orgenome edit, at least some of which include at least one genetic orepigenetic modification, is provided by the method; related aspectsinclude a plant having a phenotype of interest associated with thegenetic or epigenetic modification, provided by either regeneration of aplant having the phenotype of interest from a plant cell or plantprotoplast selected from the heterogeneous population of plant cellshaving a target gene or genome edit, or by selection of a plant havingthe phenotype of interest from a heterogeneous population of plantsgrown or regenerated from the population of plant cells having a targetgene edit or genome edit. Examples of phenotypes of interest includeherbicide resistance, improved tolerance of abiotic stress (e.g.,tolerance of temperature extremes, drought, or salt) or biotic stress(e.g., resistance to nematode, bacterial, or fungal pathogens), improvedutilization of nutrients or water, modified lipid, carbohydrate, orprotein composition, improved flavor or appearance, improved storagecharacteristics (e.g., resistance to bruising, browning, or softening),increased yield, altered morphology (e.g., floral architecture or color,plant height, branching, root structure). In an embodiment, aheterogeneous population of plant cells having a target gene edit orgenome edit (or seedlings or plants grown or regenerated therefrom) isexposed to conditions permitting expression of the phenotype ofinterest; e.g., selection for herbicide resistance can include exposingthe population of plant cells having a target gene edit or genome edit(or seedlings or plants grown or regenerated therefrom) to an amount ofherbicide or other substance that inhibits growth or is toxic, allowingidentification and selection of those resistant plant cells (orseedlings or plants) that survive treatment. Methods for obtainingregenerable plant structures and regenerating plants from plant cells orregenerable plant structures can be adapted from published procedures(Roest and Gilissen, Acta Bot. Neerl., 1989, 38(1), 1-23; Bhaskaran andSmith, Crop Sci. 30(6):1328-1337; Ikeuchi et al., Development, 2016,143: 1442-1451). Methods for obtaining regenerable plant structures andregenerating plants from plant cells or regenerable plant structures canalso be adapted from US Patent Application Publication No. 20170121722,which is incorporated herein by reference in its entirety andspecifically with respect to such disclosure. Also provided areheterogeneous populations, arrays, or libraries of such plants,succeeding generations or seeds of such plants grown or regenerated fromthe plant cells or plant protoplasts, having a target gene edit orgenome edit, parts of the plants (including plant parts used in graftingas scions or rootstocks), or products (e.g., fruits or other edibleplant parts, cleaned grains or seeds, edible oils, flours or starches,proteins, and other processed products) made from the plants or theirseeds. Embodiments include plants grown or regenerated from the plantcells having a target gene edit or genome edit, wherein the plantscontain cells or tissues that do not have a genetic or epigeneticmodification, e.g., grafted plants in which the scion or rootstockcontains a genetic or epigenetic modification, or chimeric plants inwhich some but not all cells or tissues contain a genetic or epigeneticmodification. Plants in which grafting is commonly useful include manyfruit trees and plants such as many citrus trees, apples, stone fruit(e.g., peaches, apricots, cherries, and plums), avocados, tomatoes,eggplant, cucumber, melons, watermelons, and grapes as well as variousornamental plants such as roses. Grafted plants can be grafts betweenthe same or different (generally related) species. Additional relatedaspects include a hybrid plant provided by crossing a first plant grownor regenerated from a plant cell or plant protoplast having a targetgene edit or genome edit and having at least one genetic or epigeneticmodification, with a second plant, wherein the hybrid plant contains thegenetic or epigenetic modification; also contemplated is seed producedby the hybrid plant. Also envisioned as related aspects are progeny seedand progeny plants, including hybrid seed and hybrid plants, having theregenerated plant as a parent or ancestor. The plant cells andderivative plants and seeds disclosed herein can be used for variouspurposes useful to the consumer or grower. The intact plant itself maybe desirable, e.g., plants grown as cover crops or as ornamentals. Inother embodiments, processed products are made from the plant or itsseeds, such as extracted proteins, oils, sugars, and starches,fermentation products, animal feed or human food, wood and woodproducts, pharmaceuticals, and various industrial products.

An exogenous gene transcription agent that stimulates transcription ofthe endogenous ODP2 gene and/or the endogenous WUS2 gene can be providedto a cell (e.g., a plant cell or plant protoplast) by any suitabletechnique. In certain embodiments, the exogenous gene transcriptionagent is provided by directly contacting a plant cell with the exogenousgene transcription agent or the polynucleotide that encodes theexogenous gene transcription agent. In certain embodiments, theexogenous gene transcription agent is provided by transporting theexogenous gene transcription agent or a polynucleotide that encodesexogenous gene transcription agent into a plant cell or plant protoplastusing a chemical, enzymatic, or physical agent. In certain embodiments,the exogenous gene transcription agent is provided by bacteriallymediated (e.g., Agrobacterium sp., Rhizobium sp., Sinorhizobium sp.,Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., Phyllobacteriumsp.) transfection of a plant cell or plant protoplast with apolynucleotide encoding the exogenous gene transcription agent; see,e.g., Broothaerts et al. (2005) Nature, 433:629-633. In an embodiment,the exogenous gene transcription agent is provided by transcription in aplant cell or plant protoplast of a DNA that encodes the exogenous genetranscription agent and is stably integrated in the genome of the plantcell or is provided to the plant cell or plant protoplast in the form ofa plasmid or expression vector (e.g., a viral vector) that encodes theexogenous gene transcription agent. In certain embodiments, theexogenous gene transcription agent is provided to the plant cell orplant protoplast as a polynucleotide that encodes exogenous genetranscription agent, e.g., in the form of an RNA (e.g., mRNA or RNAcontaining an internal ribosome entry site (IRES)) encoding theexogenous gene transcription agent. Genome altering reagents can also beintroduced into the plant cells by similar techniques.

Transient expression of an exogenous gene transcription agent thatstimulates transcription of the endogenous ODP2 gene and/or theendogenous WUS2 gene (e.g., expression of an guide RNA from a DNA, orexpression and translation of an ATF or RNA-guided DNA bindingpolypeptide from a DNA encoding the ATF or polypeptide), can be achievedby a variety of techniques. Certain embodiments are useful ineffectuating transient expression of the endogenous ODP2 and/or WUS2gene without remnants of the exogenous gene transcription agents thatprovide for the transient expression or selective genetic markersoccurring in progeny. In certain embodiments, the exogenous genetranscription agents are provided directly to the plant cells, systems,methods, and compositions as isolated molecules, as isolated orsemi-purified products of a cell free synthetic process (e.g., in vitrotranslation), or as isolated or semi-purified products of in acell-based synthetic process (e.g., such as in a bacterial or other celllysate). In certain embodiments, artificial transcription factors (ATFs)are targeted to the plant cell or cell nucleus in a manner that insurestransient expression (e.g., by methods adapted from Gao et al. 2016; orLi et al. 2009). In certain embodiments, the exogenous genetranscription agent is delivered into the plant cell by delivery of theagent itself in the absence of any polynucleotide that encodes theagent. Examples of exogenous gene transcription agents that can bedelivered in the absence of any encoding polynucleotides includepolypeptide ATFs (e.g., aZFPs or aTALEs), RNA-guided DNA bindingpolypeptide, and RNA guides. RNA-guided DNA binding polypeptide/RNAguides can be delivered separately and/or as RNP complexes. In certainembodiments, ATF proteins can be produced in a heterologous system,purified and delivered to plant cells by particle bombardment (e.g., bymethods adapted from Martin-Ortigosa and Wang 2014). In embodimentswhere the exogenous gene transcription agents are delivered in theabsence of any encoding polynucleotides, the delivered agent is expectedto degrade over time in the absence of ongoing expression from anyintroduced encoding polynucleotides to result in transient endogenousODP2 gene and/or the endogenous WUS2 gene expression. In certainembodiments, the exogenous gene transcription agent is delivered intothe plant cell by delivery of a polynucleotide that encodes the agent.In certain embodiments, ATFs can be encoded on a bacterial plasmid anddelivered to plant tissue by particle bombardment (e.g., by methodsadapted from Hamada et al. 2018; or Kirienko, Luo, and Sylvester 2012).In certain embodiments, ATFs can be encoded on a T-DNA and transientlytransferred to plant cells using agrobacterium (e.g., by methods adaptedfrom Leonelli et al. 2016; or Wu et al. 2014). In certain embodiments,ATFs can be encoded in a viral genome and delivered to plants (e.g., bymethods adapted from Honig et al. 2015). In certain embodiments, ATFscan be encoded in mRNA or an RNA comprising an IRES and delivered totarget plant cells. In certain embodiments where the exogenous genetranscription agent comprises an RNA-guided DNA binding polypeptide andan RNA guide, the polypeptide or guide can be delivered by a combinationof: (i) an encoding polynucleotide for either polypeptide or the guide;and (ii) either polypeptide or the guide itself in the absence of anencoding polynucleotide. In certain embodiments, the exogenous genetranscription agent is delivered into the plant cell by delivery of apolynucleotide that encodes the agent. In certain embodiments, thepolynucleotide that encodes the exogenous gene transcription agent isnot integrated into a plant cell genome (e.g., as a polynucleotidelacking sequences that provide for integration, by agroinfiltration onan integration deficient T-DNA vector or system, or in a viral vector),is not operably linked to polynucleotides which provide for autonomousreplication, and/or only provided with factors (e.g., viral replicationproteins) that provide for autonomous replication. Suitable techniquesfor transient expression including biolistic and other delivery ofpolynucleotides, agroinfiltration, and use of viral vectors disclosed byCanto, 2016 and others can be adapted for transient expression of theagents provided herein. Transient expression of the agent encoded by anon-integrated polynucleotide effectuated by excision of thepolynucleotide and/or regulated expression of the agent. In certainembodiments, the polynucleotide that encodes the exogenous genetranscription agent is integrated into a plant cell genome (e.g., anuclear or plastid genome) and transient expression of the agent iseffectuated by excision of the polynucleotide and/or regulatedexpression of the agent. Excision of a polynucleotide encoding the agentcan be provided by use of site-specific recombination systems (e.g.,Cre-Lox, FLP-FRT). Regulated expression of the agent can be effectuatedby methods including: (i) operable linkage of the polynucleotideencoding the agent to a developmentally-regulated, de-repressable,and/or inducible promoter; and/or (ii) introduction of a polynucleotide(e.g., dsRNA or amiRNA) that can induce siRNA-mediated inhibition of theagent. Suitable site-specific recombination systems as well asdevelopmentally-regulated, de-repressable, and/or inducible promotersinclude those disclosed in US Patent Application Publication No.20170121722, which is incorporated herein by reference in its entiretyand specifically with respect to such disclosure. In any of theaforementioned embodiments, transient expression of the endogenous ODP2and/or WUS2 genes can also be achieved by using an exogenous genetranscription agent comprising a DNA binding domain or complex and atranscription activation domain (TAD) that can be operably associatedthrough binding a common ligand (e.g., the iDimerize™ RegulatedTranscription System that uses Dmr A, B, or C dimerization domains andligands; Takara Bio, USA, Inc.). In such embodiments, transientexpression of the endogenous ODP2 and/or WUS2 genes can occur uponaddition of the common ligand.

Polynucleotides that can be used to effectuate transient expression ofan exogenous gene transcription agent (e.g., a polynucleotide encodingan ATF, RNA-guided DNA binding polypeptide, and/or a guide RNA) include:(a) double-stranded RNA; (b) single-stranded RNA; (c) chemicallymodified RNA; (d) double-stranded DNA; (e) single-stranded DNA; (f)chemically modified DNA; or (g) a combination of (a)-(f). Certainembodiments of the polynucleotide further include additional nucleotidesequences that provide useful functionality; non-limiting examples ofsuch additional nucleotide sequences include an aptamer or riboswitchsequence, nucleotide sequence that provides secondary structure such asstem-loops or that provides a sequence-specific site for an enzyme(e.g., a sequence-specific recombinase or endonuclease site), T-DNA(e.g., DNA sequence encoding an exogenous gene transcription agent isenclosed between left and right T-DNA borders from Agrobacterium spp. orfrom other bacteria that infect or induce tumors in plants), a DNAnuclear-targeting sequence, a regulatory sequence such as a promotersequence, and a transcript-stabilizing or -destabilizing sequence.Certain embodiments of the polynucleotide include those wherein thepolynucleotide is complexed with, or covalently or non-covalently boundto, a non-nucleic acid element, e.g., a carrier molecule, an antibody,an antigen, a viral movement protein, a cell-penetrating or pore-formingpeptide, a polymer, a detectable label, a quantum dot, or a particulateor nanoparticulate.

Various treatments are useful in delivery of an exogenous genetranscription agent that stimulates transcription of the endogenous ODP2gene and/or the endogenous WUS2 gene to a plant cell. In certainembodiments, one or more treatments is employed to deliver the agent(e.g., comprising a polynucleotide, polypeptide or combination thereof)into a plant cell or plant protoplast, e.g., through barriers such as acell wall, a plasma membrane, a nuclear envelope, and/or other lipidbilayer. In certain embodiments, a polynucleotide-, polypeptide-, orRNP-containing composition comprising the agent(s) are delivereddirectly, for example by direct contact of the composition with a plantcell. Aforementioned compositions can be provided in the form of aliquid, a solution, a suspension, an emulsion, a reverse emulsion, acolloid, a dispersion, a gel, liposomes, micelles, an injectablematerial, an aerosol, a solid, a powder, a particulate, a nanoparticle,or a combination thereof can be applied directly to a plant, plant part,plant cell, or plant explant (e.g., through abrasion or puncture orotherwise disruption of the cell wall or cell membrane, by spraying ordipping or soaking or otherwise directly contacting, by microinjection).For example, a plant cell or plant protoplast is soaked in a liquidexogenous gene transcription agent-containing composition, whereby theagent is delivered to the plant cell. In certain embodiments, theagent-containing composition is delivered using negative or positivepressure, for example, using vacuum infiltration or application ofhydrodynamic or fluid pressure. In certain embodiments, theagent-containing composition is introduced into a plant cell or plantprotoplast, e.g., by microinjection or by disruption or deformation ofthe cell wall or cell membrane, for example by physical treatments suchas by application of negative or positive pressure, shear forces, ortreatment with a chemical or physical delivery agent such assurfactants, liposomes, or nanoparticles; see, e.g., delivery ofmaterials to cells employing microfluidic flow through a cell-deformingconstriction as described in US Published Patent Application2014/0287509, incorporated by reference in its entirety herein. Othertechniques useful for delivering the agent-containing composition to aplant cell or plant protoplast include: ultrasound or sonication;vibration, friction, shear stress, vortexing, cavitation; centrifugationor application of mechanical force; mechanical cell wall or cellmembrane deformation or breakage; enzymatic cell wall or cell membranebreakage or permeabilization; abrasion or mechanical scarification(e.g., abrasion with carborundum or other particulate abrasive orscarification with a file or sandpaper) or chemical scarification (e.g.,treatment with an acid or caustic agent); and electroporation. Incertain embodiments, the agent-containing composition is provided bybacterially mediated (e.g., Agrobacterium sp., Rhizobium sp.,Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp.,Phyllobacterium sp.) transfection of the plant cell or plant protoplastwith a polynucleotide encoding the agent (e.g., ATF, RNA guided ATF,and/or guide RNA); see, e.g., Broothaerts et al. (2005) Nature,433:629-633. Any of these techniques or a combination thereof arealternatively employed on the plant explant, plant part or tissue orintact plant (or seed) from which a plant cell is optionallysubsequently obtained or isolated; in certain embodiments, theagent-containing composition is delivered in a separate step after theplant cell has been isolated. In certain embodiments, the aforementionedmethods can also be used to introduce a genome altering reagent into theplant cell.

In embodiments, a treatment employed in delivery of a exogenous genetranscription agent that stimulates transcription of the endogenous ODP2gene and/or the endogenous WUS2 gene to a plant cell is carried outunder a specific thermal regime, which can involve one or moreappropriate temperatures, e.g., chilling or cold stress (exposure totemperatures below that at which normal plant growth occurs), or heatingor heat stress (exposure to temperatures above that at which normalplant growth occurs), or treating at a combination of differenttemperatures. In certain embodiments, a specific thermal regime iscarried out on the plant cell, or on a plant, plant explant, or plantpart from which a plant cell or plant protoplast is subsequentlyobtained or isolated, in one or more steps separate from the agentdelivery. In certain embodiments, the aforementioned methods can also beused to introduce a genome altering reagent into the plant cell.

In certain embodiments of the plant parts, systems, methods, andcompositions provided herein, a whole plant or plant part or seed, or anisolated plant cell, a plant explant, or the plant or plant part fromwhich a plant cell or plant protoplast is obtained or isolated, istreated with one or more delivery agents which can include at least onechemical, enzymatic, or physical agent, or a combination thereof. Incertain embodiments, an exogenous gene transcription agent thatstimulates transcription of the endogenous ODP2 gene and/or theendogenous WUS2 gene further includes one or more than one chemical,enzymatic, or physical agents for delivery. Treatment with the chemical,enzymatic or physical agent can be carried out simultaneously with theagent delivery or in one or more separate steps that precede or followthe agent delivery. In certain embodiments, a chemical, enzymatic, orphysical agent, or a combination of these, is associated or complexedwith the polynucleotide composition, with the donor templatepolynucleotide, with the exogenous gene transcription agent; examples ofsuch associations or complexes include those involving non-covalentinteractions (e.g., ionic or electrostatic interactions, hydrophobic orhydrophilic interactions, formation of liposomes, micelles, or otherheterogeneous composition) and covalent interactions (e.g., peptidebonds, bonds formed using cross-linking agents). In non-limitingexamples, the exogenous gene transcription agent is provided as aliposomal complex with a cationic lipid; the exogenous genetranscription agent is provided as a complex with a carbon nanotube;and/or exogenous gene transcription agent is provided as a fusionprotein between the agent and a cell-penetrating peptide. Examples ofagents useful for delivering the exogenous gene transcription agent(s)include the various cationic liposomes and polymer nanoparticlesreviewed by Zhang et al. (2007) J. Controlled Release, 123:1-10, and thecross-linked multilamellar liposomes described in US Patent ApplicationPublication 2014/0356414 A1, incorporated by reference in its entiretyherein. In any of the aforementioned embodiments, it is furthercontemplated that the aforementioned methods can also be used tointroduce a genome altering reagent into the plant cell.

In certain embodiments, the chemical agent used to deliver an exogenousgene transcription agent that stimulates transcription of the endogenousODP2 gene and/or the endogenous WUS2 gene can comprise:

(a) solvents (e.g., water, dimethylsulfoxide, dimethylformamide,acetonitrile, N-pyrrolidine, pyridine, hexamethylphosphoramide,alcohols, alkanes, alkenes, dioxanes, polyethylene glycol, and othersolvents miscible or emulsifiable with water or that will dissolvephosphonucleotides in non-aqueous systems);

(b) fluorocarbons (e.g., perfluorodecalin, perfluoromethyldecalin);

(c) glycols or polyols (e.g., propylene glycol, polyethylene glycol);

(d) surfactants, including cationic surfactants, anionic surfactants,non-ionic surfactants, and amphiphilic surfactants, e.g., alkyl or arylsulfates, phosphates, sulfonates, or carboxylates; primary, secondary,or tertiary amines; quaternary ammonium salts; sultaines, betaines;cationic lipids; phospholipids; tallowamine; bile acids such as cholicacid; long chain alcohols; organosilicone surfactants including nonionicorganosilicone surfactants such as trisiloxane ethoxylate surfactants ora silicone polyether copolymer such as a copolymer of polyalkylene oxidemodified heptamethyl trisiloxane and allyloxypolypropylene glycolmethylether (commercially available as SILWET L-77™ brand surfactanthaving CAS Number 27306-78-1 and EPA Number CAL. REG. NO. 5905-50073-AA,Momentive Performance Materials, Inc., Albany, N.Y.); specific examplesof useful surfactants include sodium lauryl sulfate, the Tween series ofsurfactants, Triton-X100, Triton-X114, CHAPS and CHAPSO, Tergitol-typeNP-40, Nonidet P-40;

(e) lipids, lipoproteins, lipopolysaccharides;

(f) acids, bases, caustic agents;

(g) peptides, proteins, or enzymes (e.g., cellulase, pectolyase,maceroenzyme, pectinase), including cell-penetrating or pore-formingpeptides (e.g., (BO100)2K8, Genscript; poly-lysine, poly-arginine, orpoly-homoarginine peptides; gamma zein, see US Patent Applicationpublication 2011/0247100, incorporated herein by reference in itsentirety; transcription activator of human immunodeficiency virus type 1(“HIV-1 Tat”) and other Tat proteins, see, e.g.,www[dot]lifetein[dot]com/Cell_Penetrating_Peptides[dot]html and Jarver(2012) Mol. Therapy—Nucleic Acids, 1:e27,1-17); octa-arginine ornona-arginine; poly-homoarginine (see Unnamalai et al. (2004) FEBSLetters, 566:307-310); see also the database of cell-penetratingpeptides CPPsite 2.0 publicly available atcrdd[dot]osdd[dot]net/raghava/cppsite/

(h) RNase inhibitors;

(i) cationic branched or linear polymers such as chitosan, poly-lysine,DEAE-dextran, polyvinylpyrrolidone (“PVP”), or polyethylenimine (“PEI”,e.g., PEI, branched, MW 25,000, CAS#9002-98-6; PEI, linear, MW 5000,CAS#9002-98-6; PEI linear, MW 2500, CAS#9002-98-6);

(j) dendrimers (see, e.g., US Patent Application Publication2011/0093982, incorporated herein by reference in its entirety);

(k) counter-ions, amines or polyamines (e.g., spermine, spermidine,putrescine), osmolytes, buffers, and salts (e.g., calcium phosphate,ammonium phosphate);

(l) polynucleotides (e.g., non-specific double-stranded DNA, salmonsperm DNA);

(m) transfection agents (e.g., Lipofectin®, Lipofectamine®, andOligofectamine®, and Invivofectamine® (all from Thermo FisherScientific, Waltham, Mass.), PepFect (see Ezzat et al. (2011) NucleicAcids Res., 39:5284-5298), Transit® transfection reagents (Minis Bio,LLC, Madison, Wis.), and poly-lysine, poly-homoarginine, andpoly-arginine molecules including octo-arginine and nono-arginine asdescribed in Lu et al. (2010) J. Agric. Food Chem., 58:2288-2294);

(n) antibiotics, including non-specific DNA double-strand-break-inducingagents (e.g., phleomycin, bleomycin, talisomycin); and/or

(o) antioxidants (e.g., glutathione, dithiothreitol, ascorbate).

In any of the aforementioned embodiments, it is further contemplatedthat the aforementioned chemical agents can also be used to introduce agenome altering reagent into the plant cell.

In certain embodiments, the chemical agent is provided simultaneouslywith the exogenous gene transcription agent that stimulatestranscription of the endogenous ODP2 gene and/or the endogenous WUS2gene. In certain embodiments, exogenous gene transcription agent iscovalently or non-covalently linked or complexed with one or morechemical agents; for example, an ATF or RNA guided DNA binding proteincan be covalently linked to a peptide or protein (e.g., acell-penetrating peptide or a pore-forming peptide) or non-covalentlycomplexed with cationic lipids, polycations (e.g., polyamines), orcationic polymers (e.g., PEI). In certain embodiments, the exogenousgene transcription agent is complexed with one or more chemical agentsto form, e.g., a solution, liposome, micelle, emulsion, reverseemulsion, suspension, colloid, or gel. In any of the aforementionedembodiments, it is further contemplated that genome altering reagentscomprising polynucleotides and/or polypeptides can be also be deliveredas described above.

In certain embodiments, the physical agent for delivery of an exogenousgene transcription agent that stimulates transcription of the endogenousODP2 gene and/or the endogenous WUS2 gene is at least one selected fromthe group consisting of particles or nanoparticles (e.g., particles ornanoparticles made of materials such as carbon, silicon, siliconcarbide, gold, tungsten, polymers, or ceramics) in various size rangesand shapes, magnetic particles or nanoparticles (e.g., silenceMagMagnetotransfection™ agent, OZ Biosciences, San Diego, Calif.), abrasiveor scarifying agents, needles or microneedles, matrices, and grids. Incertain embodiments, particulates and nanoparticulates are useful indelivery of the exogenous gene transcription agent. Useful particulatesand nanoparticles include those made of metals (e.g., gold, silver,tungsten, iron, cerium), ceramics (e.g., aluminum oxide, siliconcarbide, silicon nitride, tungsten carbide), polymers (e.g.,polystyrene, polydiacetylene, and poly(3,4-ethylenedioxythiophene)hydrate), semiconductors (e.g., quantum dots), silicon (e.g., siliconcarbide), carbon (e.g., graphite, graphene, graphene oxide, or carbonnanosheets, nanocomplexes, or nanotubes), and composites (e.g.,polyvinylcarbazole/graphene, polystyrene/graphene, platinum/graphene,palladium/graphene nanocomposites). In certain embodiments, suchparticulates and nanoparticulates are further covalently ornon-covalently functionalized, or further include modifiers orcross-linked materials such as polymers (e.g., linear or branchedpolyethylenimine, poly-lysine), polynucleotides (e.g., DNA or RNA),polysaccharides, lipids, polyglycols (e.g., polyethylene glycol,thiolated polyethylene glycol), polypeptides or proteins, and detectablelabels (e.g., a fluorophore, an antigen, an antibody, or a quantum dot).In various embodiments, such particulates and nanoparticles are neutral,or carry a positive charge, or carry a negative charge. Embodiments ofcompositions including particulates include those formulated, e.g., asliquids, colloids, dispersions, suspensions, aerosols, gels, and solids.Embodiments include nanoparticles affixed to a surface or support, e.g.,an array of carbon nanotubes vertically aligned on a silicon or copperwafer substrate. Embodiments include polynucleotide compositionsincluding particulates (e.g., gold or tungsten or magnetic particles)delivered by a Biolistic-type technique or with magnetic force. The sizeof the particles used in Biolistics is generally in the “microparticle”range, for example, gold microcarriers in the 0.6, 1.0, and 1.6micrometer size ranges (see, e.g., instruction manual for the Helios®Gene Gun System, Bio-Rad, Hercules, Calif.; Randolph-Anderson et al.(2015) “Sub-micron gold particles are superior to larger particles forefficient Biolistic® transformation of organelles and some cell types”,Bio-Rad US/EG Bulletin 2015), but successful Biolistics delivery usinglarger (40 nanometer) nanoparticles has been reported in cultured animalcells; see O'Brian and Lummis (2011) BMC Biotechnol., 11:66-71. Otherembodiments of useful particulates are nanoparticles, which aregenerally in the nanometer (nm) size range or less than 1 micrometer,e.g., with a diameter of less than about 1 nm, less than about 3 nm,less than about 5 nm, less than about 10 nm, less than about 20 nm, lessthan about 40 nm, less than about 60 nm, less than about 80 nm, and lessthan about 100 nm. Specific, non-limiting embodiments of nanoparticlescommercially available (all from Sigma-Aldrich Corp., St. Louis, Mo.)include gold nanoparticles with diameters of 5, 10, or 15 nm; silvernanoparticles with particle sizes of 10, 20, 40, 60, or 100 nm;palladium “nanopowder” of less than 25 nm particle size; single-,double-, and multi-walled carbon nanotubes, e.g., with diameters of0.7-1.1, 1.3-2.3, 0.7-0.9, or 0.7-1.3 nm, or with nanotube bundledimensions of 2-10 nm by 1-5 micrometers, 6-9 nm by 5 micrometers, 7-15nm by 0.5-10 micrometers, 7-12 nm by 0.5-10 micrometers, 110-170 nm by5-9 micrometers, 6-13 nm by 2.5-20 micrometers. In certain embodiments,physical agents for delivery of an exogenous gene transcription agentscan include materials such as gold, silicon, cerium, or carbon, e.g.,gold or gold-coated nanoparticles, silicon carbide whiskers,carborundum, porous silica nanoparticles, gelatin/silica nanoparticles,nanoceria or cerium oxide nanoparticles (CNPs), carbon nanotubes (CNTs)such as single-, double-, or multi-walled carbon nanotubes and theirchemically functionalized versions (e.g., carbon nanotubesfunctionalized with amide, amino, carboxylic acid, sulfonic acid, orpolyethylene glycol moeities), and graphene or graphene oxide orgraphene complexes. Such physical agents that can be adapted fordelivery of exogenous gene transcription agents include those disclosedin Wong et al. (2016) Nano Lett., 16:1161-1172; Giraldo et al. (2014)Nature Materials, 13:400-409; Shen et al. (2012) Theranostics,2:283-294; Kim et al. (2011) Bioconjugate Chem., 22:2558-2567; Wang etal. (2010) J. Am. Chem. Soc. Comm., 132:9274-9276; Zhao et al. (2016)Nanoscale Res. Lett., 11:195-203; and Choi et al. (2016) J. ControlledRelease, 235:222-235. See also, for example, the various types ofparticles and nanoparticles, their preparation, and methods for theiruse, e.g., in delivering polynucleotides and polypeptides to cells,disclosed in US Patent Application Publications 2010/0311168,2012/0023619, 2012/0244569, 2013/0145488, 2013/0185823, 2014/0096284,2015/0040268, 2015/0047074, and 2015/0208663, all of which areincorporated herein by reference in their entirety. In any of theaforementioned embodiments, it is further contemplated that genomealtering reagents comprising polynucleotides and/or polypeptides can bealso be delivered as described above.

In certain embodiments wherein the exogenous gene transcription agentscomprise a gRNA (or polynucleotide encoding the gRNA) is provided in acomposition that further includes an RNA guided DNA binding polypeptidethat is nuclease activity deficient (or a polynucleotide that encodesthe same), one or more one chemical, enzymatic, or physical agent cansimilarly be employed. In certain embodiments, the RNA guide and thenuclease activity deficient RNA-guided DNA binding polypeptide (ndRGDBP)or polynucleotide encoding the same) are provided separately, e.g., in aseparate composition. Such compositions can include other chemical orphysical agents (e.g., solvents, surfactants, proteins or enzymes,transfection agents, particulates or nanoparticulates), such as thosedescribed above as useful in the polynucleotide compositions. Forexample, porous silica nanoparticles are useful for delivering a DNArecombinase into maize cells; see, e.g., Martin-Ortigosa et al. (2015)Plant Physiol., 164:537-547, and can be adapted to providing a ndRGDBPor polynucleotide encoding the same into a maize or other plant cell. Inone embodiment, the polynucleotide composition includes a gRNA and thendRGDBP, and further includes a surfactant and a cell-penetratingpeptide (CPP) which can be operably linked to the ndRGDBP. In anembodiment, the polynucleotide composition includes a plasmid or viralvector that encodes both the gRNA and the ndRGDBP, and further includesa surfactant and carbon nanotubes. In an embodiment, the polynucleotidecomposition includes multiple gRNAs and an mRNA encoding the ndRGDBP,and further includes particles (e.g., gold or tungsten particles), andthe polynucleotide composition is delivered to a plant cell or plantprotoplast by Biolistics. In any of the aforementioned embodiments, itis further contemplated that other polynucleotides of interest includinggenome altering reagents can also be delivered before, during, or afterdelivery of the gRNA and the ndRGDBP.

In certain embodiments, the plant, plant explant, or plant part fromwhich a plant cell is obtained or isolated is treated with one or morechemical, enzymatic, or physical agent(s) in the process of obtaining,isolating, or treating the plant cell. In certain embodiments, the plantcell, plant, plant explant, or plant part is treated with an abrasive, acaustic agent, a surfactant such as Silwet L-77 or a cationic lipid, oran enzyme such as cellulase. In any of the aforementioned embodiments,it is further contemplated that other polynucleotides of interestincluding genome altering reagents can also be delivered before, during,or after delivery of the endogenous gene transcription agents.

In certain embodiments, one or more than one chemical, enzymatic, orphysical agent, separately or in combination with the polynucleotidecomposition encoding the exogenous gene transcription agent thatstimulates transcription of the endogenous ODP2 gene and/or theendogenous WUS2 gene, is provided/applied at a location in the plant orplant part other than the plant location, part, or tissue from which theplant cell is treated, obtained, or isolated. In certain embodiments,the polynucleotide composition is applied to adjacent or distal cells ortissues and is transported (e.g., through the vascular system or bycell-to-cell movement) to the meristem from which plant cells aresubsequently isolated. In certain embodiments, thepolynucleotide-containing composition is applied by soaking a seed orseed fragment or zygotic or somatic embryo in thepolynucleotide-containing composition, whereby the polynucleotide isdelivered to the plant cell. In certain embodiments, a flower bud orshoot tip is contacted with a polynucleotide-containing composition,whereby the polynucleotide is delivered to cells in the flower bud orshoot tip from which desired plant cells (e.g., plant cells subjected toa transient increase in expression of the endogenous ODP2 gene and/orthe endogenous WUS2 gene) are obtained. In certain embodiments, apolynucleotide-containing composition is applied to the surface of aplant or of a part of a plant (e.g., a leaf surface), whereby thepolynucleotide(s) are delivered to tissues of the plant from whichdesired plant cells are obtained. In certain embodiments a whole plantor plant tissue is subjected to particle- or nanoparticle-mediateddelivery (e.g., Biolistics or carbon nanotube or nanoparticle delivery)of a polynucleotide-containing composition, whereby thepolynucleotide(s) are delivered to cells or tissues from which plantcells are subsequently obtained. In any of the aforementionedembodiments, it is further contemplated that other polynucleotides ofinterest including genome altering reagents can also be deliveredbefore, during, or after delivery of the endogenous gene transcriptionagents.

Genome altering reagents include gene editing molecules for inducing agenetic modification in the plant cells having improved plant cellregenerative potential provided herein. In certain embodiments, suchgenome altering reagents can include: (i) a polynucleotide selected fromthe group consisting of an RNA guide for an RNA-guided nuclease, a DNAencoding an RNA guide for an RNA-guided nuclease; (ii) a nucleaseselected from the group consisting of an RNA-guided nuclease, anRNA-guided DNA endonuclease, a type II Cas nuclease, a Cas9, a type VCas nuclease, a Cpf1, a CasY, a CasX, a C2c1, a C2c3, an engineerednuclease, a codon-optimized nuclease, a zinc-finger nuclease (ZFN), atranscription activator-like effector nuclease (TAL-effector nuclease),Argonaute, a meganuclease or engineered meganuclease; (iii) apolynucleotide encoding one or more nucleases capable of effectuatingsite-specific modification of a target nucleotide sequence; and/or (iv)a donor template polynucleotide. In certain embodiments, at least onedelivery agent is selected from the group consisting of solvents,fluorocarbons, glycols or polyols, surfactants; primary, secondary, ortertiary amines and quaternary ammonium salts; organosiliconesurfactants; lipids, lipoproteins, lipopolysaccharides; acids, bases,caustic agents; peptides, proteins, or enzymes; cell-penetratingpeptides; RNase inhibitors; cationic branched or linear polymers;dendrimers; counter-ions, amines or polyamines, osmolytes, buffers, andsalts; polynucleotides; transfection agents; antibiotics; chelatingagents such as ammonium oxalate, EDTA, EGTA, or cyclohexane diaminetetraacetate, non-specific DNA double-strand-break-inducing agents; andantioxidants; particles or nanoparticles, magnetic particles ornanoparticles, abrasive or scarifying agents, needles or microneedles,matrices, and grids. In certain embodiments, the plant cell, system,method, or composition comprising the plant cells provided hereinfurther includes (a) at least one plant cell having a Cas9, a Cpf1, aCasY, a CasX, a C2c1, or a C2c3 nuclease; (b) at least one guide RNA;and (c) optionally, at least one chemical, enzymatic, or physicaldelivery agent.

Gene editing molecules of use in the systems, methods, compositions, andreaction mixtures provided herein include molecules capable ofintroducing a double-strand break (“DSB”) in double-stranded DNA, suchas in genomic DNA or in a target gene located within the genomic DNA aswell as accompanying guide RNA or donor template polynucleotides.Examples of such gene editing molecules include: (a) a nuclease selectedfrom the group consisting of an RNA-guided nuclease, an RNA-guided DNAendonuclease, a type II Cas nuclease, a Cas9, a type V Cas nuclease, aCpf1, a CasY, a CasX, a C2c1, a C2c3, an engineered nuclease, acodon-optimized nuclease, a zinc-finger nuclease (ZFN), a transcriptionactivator-like effector nuclease (TAL-effector nuclease), an Argonaute,and a meganuclease or engineered meganuclease; (b) a polynucleotideencoding one or more nucleases capable of effectuating site-specificalteration (such as introduction of a DSB) of a target nucleotidesequence; (c) a guide RNA (gRNA) for an RNA-guided nuclease, or a DNAencoding a gRNA for an RNA-guided nuclease; and (d) donor templatepolynucleotides.

CRISPR-type genome editing can be adapted for use in the plant cells,systems, methods, and compositions provided herein in several ways.CRISPR elements, i.e., gene editing molecules comprising CRISPRendonucleases and CRISPR single-guide RNAs or polynucleotides encodingthe same, are useful in effectuating genome editing without remnants ofthe CRISPR elements or selective genetic markers occurring in progeny.In certain embodiments, the CRISPR elements are provided directly to theplant cells, systems, methods, and compositions as isolated molecules,as isolated or semi-purified products of a cell free synthetic process(e.g., in vitro translation), or as isolated or semi-purified productsof in a cell-based synthetic process (e.g., such as in a bacterial orother cell lysate). In certain embodiments, genome-inserted CRISPRelements are useful in plant lines adapted for use in the systems,methods, and compositions provide herein. In certain embodiments, plantsor plant cells used in the systems, methods, and compositions providedherein can comprise a transgene that expresses a CRISPR endonuclease(e.g., a Cas9, a Cpf1-type or other CRISPR endonuclease). In certainembodiments, one or more CRISPR endonucleases with unique PAMrecognition sites can be used. Cpf1 endonuclease and corresponding guideRNAs and PAM sites are disclosed in US Patent Application Publication2016/0208243 A1, which is incorporated herein by reference for itsdisclosure of DNA encoding Cpf1 endonucleases and guide RNAs and PAMsites. Introduction of one or more of a wide variety of CRISPR guideRNAs that interact with CRISPR endonucleases integrated into a plantgenome or otherwise provided to a plant is useful for genetic editingfor providing desired phenotypes or traits, for trait screening, or forgene editing mediated trait introgression (e.g., for introducing a traitinto a new genotype without backcrossing to a recurrent parent or withlimited backcrossing to a recurrent parent). Multiple endonucleases canbe provided in expression cassettes with the appropriate promoters toallow multiple genome editing in a spatially or temporally separatedfashion in either in chromosome DNA or episome DNA.

CRISPR technology for editing the genes of eukaryotes is disclosed in USPatent Application Publications 2016/0138008A1 and US2015/0344912A1, andin U.S. Pat. Nos. 8,697,359, 8,771,945, 8,945,839, 8,999,641, 8,993,233,8,895,308, 8,865,406, 8,889,418, 8,871,445, 8,889,356, 8,932,814,8,795,965, and 8,906,616. Cpf1 endonuclease and corresponding guide RNAsand PAM sites are disclosed in US Patent Application Publication2016/0208243 A1. Other CRISPR nucleases useful for editing genomesinclude C2c1 and C2c3 (see Shmakov et al. (2015) Mol. Cell, 60:385-397)and CasX and CasY (see Burstein et al. (2016) Nature,doi:10.1038/nature21059). Plant RNA promoters for expressing CRISPRguide RNA and plant codon-optimized CRISPR Cas9 endonuclease aredisclosed in International Patent Application PCT/US2015/018104(published as WO 2015/131101 and claiming priority to U.S. ProvisionalPatent Application 61/945,700). Methods of using CRISPR technology forgenome editing in plants are disclosed in US Patent ApplicationPublications US 2015/0082478A1 and US 2015/0059010A1 and inInternational Patent Application PCT/US2015/038767 A1 (published as WO2016/007347 and claiming priority to U.S. Provisional Patent Application62/023,246). All of the patent publications referenced in this paragraphare incorporated herein by reference in their entirety.

For the purposes of gene editing, CRISPR arrays can be designed tocontain one or multiple guide RNA sequences corresponding to a desiredtarget DNA sequence; see, for example, Cong et al. (2013) Science,339:819-823; Ran et al. (2013) Nature Protocols, 8:2281-2308. At least16 or 17 nucleotides of gRNA sequence are required by Cas9 for DNAcleavage to occur; for Cpf1 at least 16 nucleotides of gRNA sequence areneeded to achieve detectable DNA cleavage and at least 18 nucleotides ofgRNA sequence were reported necessary for efficient DNA cleavage invitro; see Zetsche et al. (2015) Cell, 163:759-771. In practice, guideRNA sequences are generally designed to have a length of 17-24nucleotides (frequently 19, 20, or 21 nucleotides) and exactcomplementarity (i.e., perfect base-pairing) to the targeted gene ornucleic acid sequence; guide RNAs having less than 100% complementarityto the target sequence can be used (e.g., a gRNA with a length of 20nucleotides and 1-4 mismatches to the target sequence) but can increasethe potential for off-target effects. The design of effective guide RNAsfor use in plant genome editing is disclosed in US Patent ApplicationPublication 2015/0082478 A1, the entire specification of which isincorporated herein by reference. More recently, efficient gene editinghas been achieved using a chimeric “single guide RNA” (“sgRNA”), anengineered (synthetic) single RNA molecule that mimics a naturallyoccurring crRNA-tracrRNA complex and contains both a tracrRNA (forbinding the nuclease) and at least one crRNA (to guide the nuclease tothe sequence targeted for editing); see, for example, Cong et al. (2013)Science, 339:819-823; Xing et al. (2014) BMC Plant Biol., 14:327-340.Chemically modified sgRNAs have been demonstrated to be effective ingenome editing; see, for example, Hendel et al. (2015) NatureBiotechnol., 985-991. The design of effective gRNAs for use in plantgenome editing is disclosed in US Patent Application Publication2015/0082478 A1, the entire specification of which is incorporatedherein by reference.

Other nucleases capable of effecting site-specific modification of atarget nucleotide sequence in the systems, methods, and compositionsprovided herein include zinc-finger nucleases (ZFNs), transcriptionactivator-like effector nucleases (TAL-effector nucleases or TALENs),Argonaute proteins, and a meganuclease or engineered meganuclease. Zincfinger nucleases (ZFNs) are engineered proteins comprising a zinc fingerDNA-binding domain fused to a nucleic acid cleavage domain, e.g., anuclease. The zinc finger binding domains provide specificity and can beengineered to specifically recognize any desired target DNA sequence.For a review of the construction and use of ZFNs in plants and otherorganisms, see, e.g., Urnov et al. (2010) Nature Rev. Genet.,11:636-646. The zinc finger DNA binding domains are derived from theDNA-binding domain of a large class of eukaryotic transcription factorscalled zinc finger proteins (ZFPs). The DNA-binding domain of ZFPstypically contains a tandem array of at least three zinc “fingers” eachrecognizing a specific triplet of DNA. A number of strategies can beused to design the binding specificity of the zinc finger bindingdomain. One approach, termed “modular assembly”, relies on thefunctional autonomy of individual zinc fingers with DNA. In thisapproach, a given sequence is targeted by identifying zinc fingers foreach component triplet in the sequence and linking them into amultifinger peptide. Several alternative strategies for designing zincfinger DNA binding domains have also been developed. These methods aredesigned to accommodate the ability of zinc fingers to contactneighboring fingers as well as nucleotide bases outside their targettriplet. Typically, the engineered zinc finger DNA binding domain has anovel binding specificity, compared to a naturally-occurring zinc fingerprotein. Engineering methods include, for example, rational design andvarious types of selection. Rational design includes, for example, theuse of databases of triplet (or quadruplet) nucleotide sequences andindividual zinc finger amino acid sequences, in which each triplet orquadruplet nucleotide sequence is associated with one or more amino acidsequences of zinc fingers which bind the particular triplet orquadruplet sequence. See, e.g., U.S. Pat. Nos. 6,453,242 and 6,534,261,both incorporated herein by reference in their entirety. Exemplaryselection methods (e.g., phage display and yeast two-hybrid systems) arewell known and described in the literature. In addition, enhancement ofbinding specificity for zinc finger binding domains has been describedin U.S. Pat. No. 6,794,136, incorporated herein by reference in itsentirety. In addition, individual zinc finger domains may be linkedtogether using any suitable linker sequences. Examples of linkersequences are publicly known, e.g., see U.S. Pat. Nos. 6,479,626;6,903,185; and 7,153,949, incorporated herein by reference in theirentirety. The nucleic acid cleavage domain is non-specific and istypically a restriction endonuclease, such as Fok1. This endonucleasemust dimerize to cleave DNA. Thus, cleavage by Fok1 as part of a ZFNrequires two adjacent and independent binding events, which must occurin both the correct orientation and with appropriate spacing to permitdimer formation. The requirement for two DNA binding events enables morespecific targeting of long and potentially unique recognition sites.Fok1 variants with enhanced activities have been described; see, e.g.,Guo et al. (2010) J. Mol. Biol., 400:96-107.

Transcription activator like effectors (TALEs) are proteins secreted bycertain Xanthomonas species to modulate gene expression in host plantsand to facilitate the colonization by and survival of the bacterium.TALEs act as transcription factors and modulate expression of resistancegenes in the plants. Recent studies of TALEs have revealed the codelinking the repetitive region of TALEs with their target DNA-bindingsites. TALEs comprise a highly conserved and repetitive regionconsisting of tandem repeats of mostly 33 or 34 amino acid segments. Therepeat monomers differ from each other mainly at amino acid positions 12and 13. A strong correlation between unique pairs of amino acids atpositions 12 and 13 and the corresponding nucleotide in the TALE-bindingsite has been found. The simple relationship between amino acid sequenceand DNA recognition of the TALE binding domain allows for the design ofDNA binding domains of any desired specificity. TALEs can be linked to anon-specific DNA cleavage domain to prepare genome editing proteins,referred to as TAL-effector nucleases or TALENs. As in the case of ZFNs,a restriction endonuclease, such as Fok1, can be conveniently used. Fora description of the use of TALENs in plants, see Mahfouz et al. (2011)Proc. Natl. Acad. Sci. USA, 108:2623-2628 and Mahfouz (2011) GM Crops,2:99-103.

Argonautes are proteins that can function as sequence-specificendonucleases by binding a polynucleotide (e.g., a single-stranded DNAor single-stranded RNA) that includes sequence complementary to a targetnucleotide sequence) that guides the Argonaut to the target nucleotidesequence and effects site-specific alteration of the target nucleotidesequence; see, e.g., US Patent Application Publication 2015/0089681,incorporated herein by reference in its entirety.

In related embodiments, zinc finger nucleases, TALENs, and Argonautesare used in conjunction with other functional domains. For example, thenuclease activity of these nucleic acid targeting systems can be alteredso that the enzyme binds to but does not cleave the DNA. Examples offunctional domains include transposase domains, integrase domains,recombinase domains, resolvase domains, invertase domains, proteasedomains, DNA methyltransferase domains, DNA hydroxylmethylase domains,DNA demethylase domains, histone acetylase domains, histone deacetylasedomains, nuclease domains, repressor domains, activator domains,nuclear-localization signal domains, transcription-regulatory protein(or transcription complex recruiting) domains, cellular uptake activityassociated domains, nucleic acid binding domains, antibody presentationdomains, histone modifying enzymes, recruiter of histone modifyingenzymes; inhibitor of histone modifying enzymes, histonemethyltransferases, histone demethylases, histone kinases, histonephosphatases, histone ribosylases, histone deribosylases, histoneubiquitinases, histone deubiquitinases, histone biotinases and histonetail proteases. Non-limiting examples of functional domains include atranscriptional activation domain, a transcription repression domain,and an SHH1, SUVH2, or SUVH9 polypeptide capable of reducing expressionof a target nucleotide sequence via epigenetic modification; see, e.g.,US Patent Application Publication 2016/0017348, incorporated herein byreference in its entirety. Genomic DNA may also be modified via baseediting using a fusion between a catalytically inactive Cas9 (dCas9)fused to a cytidine deaminase which converts cytosine (C) to uridine(U), thereby effecting a C to T substitution; see Komor et al. (2016)Nature, 533:420-424. In other embodiments, adenine base editors (ABEs)can be used to convert A/T base pairs to G/C base pairs in genomic DNA(Gaudelli et al., 2017).

Other genome altering reagents used in plant cells and methods providedherein include transgenes or vectors comprising the same. Suchtransgenes can confer useful traits that include herbicide tolerance,pest tolerance (e.g., tolerance to insects, nematodes, or plantpathogenic fungi and bacteria), improved yield, increased and/orqualitatively improved oil, starch, and protein content, improvedabiotic stress tolerance (e.g., improved or enhanced water useefficiency or drought tolerance, osmotic stress tolerance, high salinitystress tolerance, heat stress tolerance, enhanced cold tolerance,including cold germination tolerance), and the like. Such transgenesinclude both transgenes that confer the trait by expression of anexogenous protein as well as transgenes that confer the trait byinhibiting expression of endogenous plant genes (e.g., by inducing ansiRNA response which inhibits expression of the endogenous plant genes).Transgenes that can provide such traits are disclosed in US PatentApplication Publication Nos. 20170121722 and 20170275636, which are eachincorporated herein by reference in their entireties and specificallywith respect to such disclosures.

In some embodiments, one or more polynucleotides or vectors drivingexpression of one or more polynucleotides encoding any of theaforementioned exogenous gene transcription agents and/or genomealtering reagents are introduced into a plant cell. In certainembodiments, a polynucleotide vector comprises a regulatory element suchas a promoter operably linked to one or more polynucleotides encodingexogenous gene transcription agents or genome altering reagents. In suchembodiments, expression of these polynucleotides can be controlled byselection of the appropriate promoter, particularly promoters functionalin a plant cell; useful promoters include constitutive, conditional,inducible, and temporally or spatially specific promoters (e.g., atissue specific promoter, a developmentally regulated promoter, or acell cycle regulated promoter). Developmentally regulated promoters thatcan be used include Phospholipid Transfer Protein (PLTP),fructose-1,6-bisphosphatase protein, NAD(P)-binding Rossmann-Foldprotein, adipocyte plasma membrane-associated protein-like protein,Rieske [2Fe-2S] iron-sulfur domain protein, chlororespiratory reduction6 protein, D-glycerate 3-kinase, chloroplastic-like protein, chlorophylla-b binding protein 7, chloroplastic-like protein,ultraviolet-B-repressible protein, Soul heme-binding family protein,Photosystem I reaction center subunit psi-N protein, and short-chaindehydrogenase/reductase protein that are disclosed in US PatentApplication Publication No. 20170121722, which is incorporated herein byreference in its entirety and specifically with respect to suchdisclosure. In certain embodiments, the promoter is operably linked tonucleotide sequences encoding multiple guide RNAs, wherein the sequencesencoding guide RNAs are separated by a cleavage site such as anucleotide sequence encoding a microRNA recognition/cleavage site or aself-cleaving ribozyme (see, e.g., Ferré-D'Amaré and Scott (2014) ColdSpring Harbor Perspectives Biol., 2:a003574). In certain embodiments,the promoter is an RNA polymerase III promoter operably linked to anucleotide sequence encoding one or more guide RNAs. In certainembodiments, the promoter operably linked to one or more polynucleotidesis a constitutive promoter that drives gene expression in plant cells.In certain embodiments, the promoter drives gene expression in thenucleus or in an organelle such as a chloroplast or mitochondrion.Examples of constitutive promoters include a CaMV 35S promoter asdisclosed in U.S. Pat. Nos. 5,858,742 and 5,322,938, a rice actinpromoter as disclosed in U.S. Pat. No. 5,641,876, a maize chloroplastaldolase promoter as disclosed in U.S. Pat. No. 7,151,204, and thenopaline synthase (NOS) and octopine synthase (OCS) promoters fromAgrobacterium tumefaciens. In certain embodiments, the promoter operablylinked to one or more polynucleotides encoding elements of agenome-editing system is a promoter from figwort mosaic virus (FMV), aRUBISCO promoter, or a pyruvate phosphate dikinase (PPDK) promoter,which is active in photosynthetic tissues. Other contemplated promotersinclude cell-specific or tissue-specific or developmentally regulatedpromoters, for example, a promoter that limits the expression of thenucleic acid targeting system to germline or reproductive cells (e.g.,promoters of genes encoding DNA ligases, recombinases, replicases, orother genes specifically expressed in germline or reproductive cells).In certain embodiments, the genome alteration is limited only to thosecells from which DNA is inherited in subsequent generations, which isadvantageous where it is desirable that expression of the genome-editingsystem be limited in order to avoid genotoxicity or other unwantedeffects. All of the patent publications referenced in this paragraph areincorporated herein by reference in their entirety.

Expression vectors or polynucleotides provided herein may contain a DNAsegment near the 3′ end of an expression cassette that acts as a signalto terminate transcription and directs polyadenylation of the resultantmRNA, and may also support promoter activity. Such a 3′ element iscommonly referred to as a “3′-untranslated region” or “3′-UTR” or a“polyadenylation signal.” In some cases, plant gene-based 3′ elements(or terminators) consist of both the 3′-UTR and downstreamnon-transcribed sequence (Nuccio et al., 2015). Useful 3′ elementsinclude: Agrobacterium tumefaciens nos 3′, tml 3′, tmr 3′, tms 3′, ocs3′, and tr7 3′ elements disclosed in U.S. Pat. No. 6,090,627,incorporated herein by reference, and 3′ elements from plant genes suchas the heat shock protein 17, ubiquitin, and fructose-1,6-biphosphatasegenes from wheat (Trilicum aestivum), and the glutelin, lactatedehydrogenase, and beta-tubulin genes from rice (Oryza sativa),disclosed in US Patent Application Publication 2002/0192813 A1,incorporated herein by reference.

In certain embodiments, a vector or polynucleotide comprising anexpression cassette includes additional components, e.g., apolynucleotide encoding a drug resistance or herbicide gene or apolynucleotide encoding a detectable marker such as green fluorescentprotein (GFP) or beta-glucuronidase (gus) to allow convenient screeningor selection of cells expressing the vector or polynucleotide.Selectable markers include genes that confer resistance to herbicidalcompounds, such as glyphosate, sulfonylureas, glufosinate ammonium,bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D).Since the transient expression of endogenous ODP2 and/or WUS2 genes canaccelerate somatic embryogenesis and embryo maturation, selectablemarker genes, selective agents, and conditions can be adjusted tominimize formation of un-edited or untransformed regenerable plantstructures (e.g., “escapes”). Such selectable marker genes and selectiveagents include the maize HRA gene (Lee et al., 1988, EMBO J 7:1241-1248)which confers resistance to sulfonylureas and imidazolinones, the CP4gene that confers resistance to glyphosate (U.S. Reissue PatentRE039247, specifically incorporated herein by reference in its entiretyand with respect to such genes and related selection methods), the GATgene which confers resistance to glyphosate (Castle et al., 2004,Science 304:1151-1154), genes that confer resistance to spectinomycinsuch as the aadA gene (Svab et al., 1990, Plant Mol Biol. 14:197-205)and the bar gene that confers resistance to glufosinate ammonium (Whiteet al., 1990, Nucl. Acids Res. 25:1062), and PAT (or moPAT for corn, seeRasco-Gaunt et al., 2003, Plant Cell Rep. 21:569-76; also see Sivamaniet al., 2019) and the PMI gene that permits growth on mannose-containingmedium (Negrotto et al., 2000, Plant Cell Rep. 22:684-690).

Embodiments

Various embodiments of the plant cells and methods provided herein areincluded in the following non-limiting list of embodiments.

1. A plant cell wherein expression of an endogenous ODP2 polypeptideand/or expression of an endogenous WUS2 polypeptide is increased incomparison to the expression of the endogenous ODP2 and/or theendogenous WUS2 polypeptides in a control plant cell, wherein the plantcell can form a regenerable plant structure, and optionally wherein theplant cell is a monocot plant cell or optionally wherein the plant cellis a maize, wheat, sorghum, or rice plant cell.

2. The plant cell of embodiment 1, wherein an exogenous polynucleotideencoding an ODP2 and/or WUS2 polypeptide is absent before and/or duringthe increase in expression of the endogenous ODP2 polypeptide and/or theendogenous WUS2 polypeptide.

3. The plant cell of embodiment 1 or 2, wherein the increase inexpression of the endogenous ODP2 polypeptide and/or expression of theendogenous WUS2 polypeptide is sufficient to increase in proliferation,somatic embryogenesis, and/or regeneration capacity of the plant cell incomparison to the control plant cell.

4. The plant cell of any one of embodiments 1, 2, or 3, wherein theincrease in expression of the endogenous ODP2 polypeptide and/orexpression of the endogenous WUS2 polypeptide is sufficient to increasein transformation efficiency and/or endogenous gene editing efficiencyof the plant cell in comparison to the control plant cell.

5. The plant cell of any one of embodiments 1-3, or 4, wherein theincrease in the expression of the endogenous ODP2 polypeptide and/orexpression of the endogenous WUS2 polypeptide is for a period of about12, 24, 30, or 36 hours to about 168 or 192 hours.

6. The plant cell of any one of embodiments 1-4, or 5, wherein said cellis located within or obtained from a cultured plant tissue explant, animmature embryo, a mature embryo, a leaf, and/or callus.

7. The plant cell of embodiment 6, wherein the plant tissue explant,embryo, or callus exhibits an increase in proliferation, somaticembryogenesis, and/or regeneration capacity in comparison to the controlplant tissue explant, embryo, or callus which was not subjected to anincrease in expression of the endogenous ODP2 polypeptide and/or theendogenous WUS2 polypeptide.

8. The plant cell of embodiment 6, wherein the plant tissue explant,embryo, or callus exhibits an increase in transformation efficiencyand/or endogenous gene editing efficiency in comparison to the controlplant tissue explant, embryo, or callus which was not subjected to anincrease in expression of the endogenous ODP2 polypeptide and/or theendogenous WUS2 polypeptide.

9. The plant cell of any one of embodiments 1 to 8 wherein the plantcell, plant tissue explant, embryo, or callus comprises inbredgermplasm, haploid germplasm, and/or a regeneration-recalcitrantgermplasm.

10. The plant cell of any one of embodiments 1 to 8, wherein the plantcell is derived from the L1 or L2 layer of an immature or mature embryo.

11. The plant cell of any one of embodiments 1 to 8, wherein the plantcell is a maize plant cell and the ODP2 polypeptide comprises an aminoacid sequence having at least 95%, 96%, 97%, or 99% amino acid sequenceidentity across the entire length of SEQ ID NO:1.

12. The plant cell of embodiment 11, wherein the endogenous ODP2polypeptide is encoded by the endogenous maize ODP2 gene located onmaize chromosome 3 and/or that is encoded by an endogenouspolynucleotide that is operably linked to an endogenous maize ODP2promoter of SEQ ID NO:3, SEQ ID NO:71, or an allelic variant thereof.

13. The plant cell of any one of embodiments 1 to 12, wherein the plantcell is a maize plant cell and the endogenous WUS2 polypeptide comprisesan amino acid sequence having at least 95%, 96%, 97%, or 99% amino acidsequence identity across the entire length of SEQ ID NO:2.

14. The plant cell of embodiment 13, wherein the endogenous WUS2polypeptide is encoded by the endogenous maize WUS2 gene located onmaize chromosome 10 and/or that is encoded by an endogenouspolynucleotide that is operably linked to an endogenous maize WUS2promoter of SEQ ID NO:4 or an allelic variant thereof.

15. The plant cell of any one of embodiments 1 to 14, wherein theexpression of the endogenous ODP2 polypeptide and/or the endogenous WUS2polypeptide is transiently increased with at least one exogenous genetranscription agent that stimulates transcription of the endogenous ODP2gene and/or with at least one exogenous gene transcription agent thatstimulates transcription of the endogenous WUS2 gene.

16. The plant cell of embodiment 15, wherein the exogenous genetranscription agent is provided as: (i) an exogenous protein; or (ii) asan exogenous protein and an exogenous guide RNA.

17. The plant cell of embodiment 16, wherein the exogenous protein ofeither (i) or (ii) is provided in the cell in the absence of anexogenous polynucleotide that encodes the protein.

18. The plant cell of embodiment 16, wherein the exogenous protein ofeither (i) or (ii) is provided in the cell by an exogenouspolynucleotide comprising a promoter that is operably linked to apolynucleotide that encodes the protein or by an exogenous RNA moleculethat encodes the protein.

19. The plant cell of embodiment 18, wherein the exogenous RNA moleculethat encodes the protein comprises an mRNA or an RNA with an internalribosome entry site (IRES).

20. The plant cell of embodiment 19, wherein the exogenouspolynucleotide or exogenous RNA are operably linked to a polynucleotidecomprising a viral vector or T-DNA in the cell.

21. The plant cell of embodiment 20, wherein the exogenous protein,exogenous polynucleotide, and/or exogenous guide RNA are in partassociated with an exogenous particle within the cell.

22. The plant cell of embodiment 15, wherein the exogenous genetranscription agent comprises: (i) a domain or complex which binds tothe promoter or 5′ untranslated region (5′ UTR) of the endogenous ODP2gene or to the promoter or 5′ UTR of the endogenous WUS2 gene; and (ii)a transcription activation domain, wherein the transcription activationdomain is operably linked or operably associated with the domain orcomplex.

23. The plant cell of embodiment 22, wherein the exogenous genetranscription agent further comprises an operably linked nuclearlocalization signal (NLS).

24. The plant cell of embodiment 22, wherein the domain that binds thepromoter or 5′ UTR comprises an artificial zinc finger (AZF) DNA bindingdomain polypeptide or an artificial transcription activator-likeeffector (TALE) DNA binding polypeptide.

25. The plant cell of embodiment 24, wherein the plant cell is a maizeplant cell and the artificial zinc finger DNA binding domain that bindsthe ODP2 promoter comprises: (i) a polypeptide having at least 85%, 90%,95%, 97%, 98%, or 99% sequence identity across the entire length of SEQID NO: 5 or 8; or (ii) a polypeptide having at one or more conservativeand/or semi-conservative amino acid substitutions in SEQ ID NO: 5 or 8.

26. The plant cell of embodiment 24, wherein the plant cell is a maizeplant cell and the artificial zinc finger DNA binding domain that bindsthe WUS2 promoter comprises: (i) a polypeptide having at least 85%, 90%,95%, 97%, 98%, or 99% sequence identity across the entire length of SEQID NO: 11 or 14; or comprises a polypeptide having at one or moreconservative and/or semi-conservative amino acid substitutions in SEQ IDNO: 11 or 14.

27. The plant cell of embodiment 24, wherein the plant cell is a maizeplant cell and the artificial TALE DNA binding polypeptide that bindsthe ODP2 promoter comprises: (i) a polypeptide having at least 85%, 90%,95%, 97%, 98%, or 99% sequence identity across the entire length of SEQID NO: 27, 81, 84, or 87; or (ii) a polypeptide having at one or moreconservative and/or semi-conservative amino acid substitutions in SEQ IDNO: 23, 25, 27, 81, 84, or 87.

28. The plant cell of embodiment 24, wherein the plant cell is a maizeplant cell and the artificial TALE DNA binding domain that binds theWUS2 promoter comprises: (i) a polypeptide having at least 85%, 90%,95%, 97%, 98%, or 99% sequence identity across the entire length of SEQID NO: 17, 19, 21, 72, 75, or 78; or (ii) a polypeptide having at one ormore conservative and/or semi-conservative amino acid substitutions inSEQ ID NO: 17, 19, 21, 72, 75, or 78.

29. The plant cell of embodiment 22, wherein the complex that binds thepromoter or 5′ UTR comprises: (i) an RNA guided DNA binding polypeptidethat is nuclease activity deficient and a guide RNA comprising about an18 or 19 to about a 21 or 22 nucleotide polynucleotide sequence which iscomplementary to a sequence immediately adjacent to a protospaceradjacent motif (PAM) in the promoter or 5′ UTR; or (ii) a 20, 21, 22,23, or 24 nucleotide polynucleotide sequence which is complementary to asequence immediately adjacent to a protospacer adjacent motif (PAM) inthe promoter or 5′ UTR.

30. The plant cell of embodiment 29, wherein the RNA guided DNA bindingpolypeptide comprises a dCAS9 polypeptide, comprises a polypeptidehaving at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity acrossthe entire length of SEQ ID NO: 29, or comprises a polypeptide having atone or more conservative and/or semi-conservative amino acidsubstitutions in SEQ ID NO: 29.

31. The plant cell of embodiment 29, wherein the RNA guided DNA bindingpolypeptide comprises; (i) a dCpf1 polypeptide;

(ii) a polypeptide having one or more conservative and/orsemi-conservative amino acid substitutions in SEQ ID NO: 44 or at least85%, 90%, 95%, 97%, 98%, or 99% sequence identity across the entirelength of SEQ ID NO: 44 and a D917A, E1006A, E1028A, D1255A, and/orN1257A amino acid substitution;

(iii) a polypeptide having one or more conservative and/orsemi-conservative amino acid substitutions in SEQ ID NO: 45 or at least85%, 90%, 95%, 97%, 98%, or 99% sequence identity across the entirelength of SEQ ID NO: 45 and a D832A, E925A, and/or D1148A amino acidsubstitution;

(iv) a polypeptide having one or more conservative and/orsemi-conservative amino acid substitutions in SEQ ID NO: 46 or at least85%, 90%, 95%, 97%, 98%, or 99% sequence identity across the entirelength of SEQ ID NO: 46 and a D917A, E1006A, E1028A, D1255A, and/orN1257A amino acid substitution; or

(v) a polypeptide having one or more conservative and/orsemi-conservative amino acid substitutions in SEQ ID NO: 47 or at least85%, 90%, 95%, 97%, 98%, or 99% sequence identity across the entirelength of SEQ ID NO: 47 and a D901A and/or E1228A amino acidsubstitution.

32. The plant cell of embodiment 29, wherein the plant cell is a maizeplant cell and the guide RNA comprises a sequence that is complementaryto either strand of an ODP2 promoter comprising SEQ ID NO: 3 or SEQ IDNO: 71 or to either strand of an WUS2 promoter comprising SEQ ID NO: 4,wherein the complementary sequence is immediately adjacent to aprotospacer adjacent motif (PAM) in SEQ ID NO: 3, 71, or 4.

33. The plant cell of embodiment 32, wherein the guide RNA comprises anRNA encoded by SEQ ID NO: 31, 32, 33, 34, or 35 that is complementary toa sequence in the endogenous maize ODP2 promoter.

34. The plant cell of embodiment 32, wherein the guide RNA comprises anRNA encoded by SEQ ID NO: 36, 37, 38, 39, or 40 that is complementary toa sequence in the endogenous maize WUS2 promoter.

35. The plant cell of any one of embodiments 1 to 34, wherein the cellcomprises at least two exogenous gene transcription agents thatstimulate transcription of the endogenous ODP2 gene and/or at least twoexogenous gene transcription agents that stimulate transcription of theendogenous WUS2 gene.

36. The plant cell of embodiment 35, wherein the exogenous genetranscription agents comprise a transcriptional activation domain thatis operably linked to or operably associated with: (i) an artificialzinc finger (AZF) DNA binding domain polypeptide; (ii) an artificialTALE DNA binding polypeptide; (iii) an RNA guided DNA bindingpolypeptide that is nuclease activity deficient and a guide RNA, or anycombination thereof.

37. The plant cell of embodiment 35, wherein the plant cell is a maizeplant cell and the exogenous gene transcription agents that stimulatetranscription of the endogenous WUS2 gene comprise a combination of atleast two artificial TALE transcription factors comprising atranscriptional activation domain and an artificial TALE DNA bindingpolypeptide of SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:72,SEQ ID NO:75, and SEQ ID NO:78.

38. The plant cell of embodiment 35, wherein the plant cell is a maizeplant cell and the exogenous gene transcription agents that stimulatetranscription of the endogenous ODP2 gene comprise a combination of atleast two distinct artificial TALE transcription factors that eachcomprise a transcriptional activation domain that is operably linked toor operably associated with an artificial TALE DNA sequence recognitiondomain polypeptide of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:81, SEQ ID NO:84, or SEQ ID NO:87.

39. The plant cell of any one of embodiments 1 to 38, wherein the cellfurther comprises a genome altering reagent.

40. The plant cell of any one of embodiments 1 to 39, wherein theregenerable plant structure comprises a somatic embryo, embryogeniccallus, somatic meristem, organogenic callus, a shoot, or a shootfurther comprising roots.

41. A method of producing a regenerable plant structure, comprising:

-   -   (i) introducing into a plant cell at least one exogenous gene        transcription agent which transiently increases expression of an        endogenous ODP2 polypeptide and/or at least one exogenous gene        transcription agent which transiently increases expression of an        endogenous WUS2 polypeptide, wherein the expression is increased        in comparison to the expression of the endogenous ODP2 and/or        the endogenous WUS2 polypeptides in a control plant cell; and,    -   (ii) culturing the plant cell to produce a regenerable plant        structure;    -   optionally wherein the plant cell is a monocot plant cell or        optionally wherein the monocot plant cell is a maize, wheat,        sorghum, or rice plant cell.

42. The method of embodiment 41, wherein an exogenous polynucleotideencoding an ODP2 and/or WUS2 polypeptide is absent before and/or duringthe transient increase in expression of the endogenous ODP2 polypeptideand/or the endogenous WUS2 polypeptide.

43. The method of embodiment 41 or 42, wherein the transient increase inexpression of the endogenous ODP2 polypeptide and/or expression of theendogenous WUS2 polypeptide is sufficient to increase proliferation,somatic embryogenesis, and/or regeneration capacity of the plant cell incomparison to the control plant cell.

44. The method of any one of embodiments 41 to 43, wherein the transientincrease in expression of the endogenous ODP2 polypeptide and/orexpression of the endogenous WUS2 polypeptide is sufficient to increasetransformation efficiency and/or endogenous gene editing efficiency incomparison to the control plant cell.

45. The method of any one of embodiments 41 to 44, wherein the transientincrease in the expression of the endogenous ODP2 polypeptide and/orexpression of the endogenous WUS2 polypeptide is for a period of about24, 30, or 36 hours to about 168 or 192 hours.

46. The method of any one of embodiments 41 to 45, wherein the exogenousgene transcription agent is introduced by electroporation, particlebombardment, transfection, Agrobacterium-mediated transformation orviral vector-mediated transfer.

47. The method of embodiment 41, wherein the exogenous genetranscription agent is introduced as: (i) an exogenous protein; or (ii)as an exogenous protein and an exogenous guide RNA, wherein said proteinand said guide RNA are optionally complexed as a RNP.

48. The method of embodiment 47, wherein the exogenous protein of either(i) or (ii) is introduced in the cell in the absence of an exogenouspolynucleotide that encodes the protein.

49. The method of embodiment 47, wherein the exogenous protein of either(i) or (ii) is introduced in the cell by an exogenous polynucleotidecomprising a promoter that is operably linked to a polynucleotide thatencodes the protein or an exogenous RNA molecule that encodes theprotein.

50. The method of embodiment 49, wherein the exogenous polynucleotide orexogenous RNA are operably linked to a polynucleotide comprising a viralvector or T-DNA in the cell.

51. The method of embodiment 49, wherein the exogenous polynucleotide isnot integrated into the nuclear or plastid genome of the plant cell.

52. The method of any one of embodiments 41 to 51, wherein said plantcell is located within or obtained from a cultured plant tissue explant,an immature embryo, a mature embryo, a leaf, and/or callus.

53. The method of embodiment 52, wherein the plant cell is derived fromthe L1 or L2 layer of an immature or mature embryo.

54. The method of any one of embodiments 41 to 53, wherein the plantcell, plant tissue explant, embryo, or callus comprises inbredgermplasm, haploid germplasm, and/or a regeneration-recalcitrantgermplasm.

55. The method of any one of embodiments 41 to 54, wherein theregenerable plant structure comprises a somatic embryo, embryogeniccallus, somatic meristem, organogenic callus, a shoot, or a shootfurther comprising roots.

56. The method of any one of embodiments 41 to 55, wherein the culturingcomprises growing of the plant cell in plant cell growth mediacomprising an auxin concentration sufficient to induce formation of asomatic embryo, embryogenic callus, somatic meristem, and/or organogeniccallus.

57. The method of embodiment 56, wherein the culturing further comprisesgrowing the somatic embryo, embryogenic callus, somatic meristem, and/ororganogenic callus in plant cell growth media comprising concentrationsof auxin and cytokinin sufficient to induce formation of a shoot.

58. The method of embodiment 57, wherein the culturing further comprisesgrowing the shoot in a plant cell growth media until the shoot formsroots.

59. The method of any one of embodiments 41 to 58, further comprisingthe step of introducing a genome altering reagent into the cell at step(i), (ii), or (i) and (ii).

60. The method of embodiment 59, wherein the genome altering reagentcomprises a transgene, a vector comprising a transgene, or genomeediting molecules.

61. The method of embodiment 60, wherein the vector comprises a T-DNA orviral vector.

62. The method of embodiment 59, wherein the genome editing moleculescomprise an RNA-guided nuclease or a polynucleotide encoding anRNA-guided nuclease, a guide RNA or a polynucleotide encoding a guideRNA, and optionally a donor template polynucleotide or a polynucleotideencoding a donor template polynucleotide.

63. The method of embodiment 62, wherein the RNA-guided nuclease andguide RNA are introduced as a ribonucleoprotein (RNP) complex.

64. The method of embodiment 59, wherein the genome editing moleculescomprise a transcription activator-like nuclease (TALEN) protein or apolynucleotide encoding a TALEN protein and optionally a donor templatepolynucleotide or a polynucleotide encoding a donor templatepolynucleotide.

65. The method of embodiment 59, wherein the genome editing moleculescomprise a zinc finger nuclease (ZnfN) protein or a polynucleotideencoding a ZnfN protein and optionally a donor template polynucleotideor a polynucleotide encoding a donor template polynucleotide.

66. The method of any one of embodiments 41 to 65, wherein the plantcell is a maize plant cell and the ODP2 polypeptide comprises an aminoacid sequence having at least 95%, 96%, 97%, or 99% amino acid sequenceidentity across the entire length of SEQ ID NO:1.

67. The method of embodiment 66, wherein the endogenous ODP2 polypeptideis encoded by the endogenous maize ODP2 gene located on maize chromosome3 and/or that is encoded by an endogenous polynucleotide that isoperably linked to an endogenous maize ODP2 promoter of SEQ ID NO:3, SEQID NO:71, or an allelic variant thereof.

68. The method of any one of embodiments 41 to 67, wherein the plantcell is a maize plant cell and the endogenous WUS2 polypeptide comprisesan amino acid sequence having at least 95%, 96%, 97%, or 99% amino acidsequence identity across the entire length of SEQ ID NO:2.

69. The method of embodiment 68, wherein the endogenous WUS2 polypeptideis encoded by the endogenous maize WUS2 gene located on maize chromosome10 and/or that is encoded by an endogenous polynucleotide that isoperably linked to an endogenous maize WUS2 promoter of SEQ ID NO:4 oran allelic variant thereof.

70. The method of any one of embodiments 41 to 69, wherein the exogenousgene transcription agent is introduced as: (i) an exogenous protein orexogenous polynucleotide encoding the protein; or (ii) as an exogenousprotein or exogenous polynucleotide encoding the protein and anexogenous guide RNA.

71. The method of embodiment 70, wherein the exogenous protein of either(i) or (ii) is introduced into the cell in the absence of an exogenouspolynucleotide that encodes the protein.

72. The method of embodiment 71, wherein the exogenous protein of either(i) or (ii) is introduced into the cell by an exogenous polynucleotidecomprising a promoter that is operably linked to a polynucleotide thatencodes the protein or by an exogenous RNA molecule that encodes theprotein.

73. The method of embodiment 72, wherein the exogenous RNA molecule thatencodes the protein comprises an mRNA or an RNA with an internalribosome entry site (IRES).

74. The method of embodiment 72, wherein the exogenous polynucleotide orexogenous RNA are operably linked to a polynucleotide comprising a viralvector or T-DNA and wherein the polynucleotide comprising a viral vectoror T-DNA is introduced into the cell.

75. The method of embodiment 70, wherein the exogenous protein,exogenous polynucleotide, and/or exogenous guide RNA are introduced intothe cell by particle bombardment of the cell.

76. The method of any one of embodiments 41 to 75, wherein the exogenousgene transcription agent comprises: (i) a domain or complex which bindsto the promoter or 5′ untranslated region (5′ UTR) of the endogenousODP2 gene or to the promoter or 5′ UTR of the endogenous WUS2 gene; and(ii) a transcription activation domain, wherein the transcriptionactivation domain is operably linked or operably associated with thedomain or complex.

77. The method of embodiment 76, wherein the exogenous genetranscription agent further comprises an operably linked nuclearlocalization signal (NLS).

78. The method of embodiment 76, wherein the domain that binds thepromoter or 5′ UTR comprises an artificial zinc finger (AZF) DNA bindingdomain polypeptide or an artificial transcription activator-likeeffector (TALE) DNA binding polypeptide.

79. The method of embodiment 78, wherein the artificial zinc finger DNAbinding domain that binds the ODP2 promoter comprises: (i) a polypeptidehaving at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity acrossthe entire length of SEQ ID NO: 5 or 8; or (ii) a polypeptide having atone or more conservative and/or semi-conservative amino acidsubstitutions in SEQ ID NO: 5 or 8.

80. The method of embodiment 78, wherein the artificial zinc finger DNAbinding domain that binds the WUS2 promoter comprises: (i) a polypeptidehaving at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity acrossthe entire length of SEQ ID NO: 11 or 14; or comprises a polypeptidehaving at one or more conservative and/or semi-conservative amino acidsubstitutions in SEQ ID NO: 11 or 14.

81. The method of embodiment 78, wherein the artificial TALE DNA bindingpolypeptide that binds the ODP2 promoter comprises: (i) a polypeptidehaving at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity acrossthe entire length of SEQ ID NO: 23, 25, 27, 81, 84, or 87; or (ii) apolypeptide having at one or more conservative and/or semi-conservativeamino acid substitutions in SEQ ID NO: 23, 25, 27, 81, 84, or 87.

82. The method of embodiment 78, wherein the artificial TALE DNA bindingdomain that binds the WUS2 promoter comprises: (i) a polypeptide havingat least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity across theentire length of SEQ ID NO: 17, 19, 21, 72, 75, or 78; or (ii) apolypeptide having at one or more conservative and/or semi-conservativeamino acid substitutions in SEQ ID NO: 17, 19, 21, 72, 75, or 78.

83. The method of embodiment 76, wherein the complex that binds thepromoter or 5′ UTR comprises: (i) an RNA guided DNA binding polypeptidethat is nuclease activity deficient and a guide RNA comprising about an18 or 19 to about a 21 or 22 nucleotide polynucleotide sequence with iscomplementary to a sequence immediately adjacent to a protospaceradjacent motif (PAM) in the promoter or 5′ UTR; or (ii) a 20, 21, 22,23, or 24 nucleotide polynucleotide sequence which is complementary to asequence immediately adjacent to a protospacer adjacent motif (PAM) inthe promoter or 5′ UTR.

84. The method of embodiment 83, wherein the RNA guided DNA bindingpolypeptide comprises a dCAS9 polypeptide, comprises a polypeptidehaving at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity acrossthe entire length of SEQ ID NO: 29, or comprises a polypeptide having atone or more conservative and/or semi-conservative amino acidsubstitutions in SEQ ID NO: 29.

85. The method of embodiment 83, wherein the RNA guided DNA bindingpolypeptide comprises:

(i) a dCpf1 polypeptide;

(ii) a polypeptide having one or more conservative and/orsemi-conservative amino acid substitutions in SEQ ID NO: 44 or at least85%, 90%, 95%, 97%, 98%, or 99% sequence identity across the entirelength of SEQ ID NO: 44 and a D917A, E1006A, E1028A, D1255A, and/orN1257A amino acid substitution;

(iii) a polypeptide having one or more conservative and/orsemi-conservative amino acid substitutions in SEQ ID NO: 45 or at least85%, 90%, 95%, 97%, 98%, or 99% sequence identity across the entirelength of SEQ ID NO: 45 and a D832A, E925A, and/or D1148A amino acidsubstitution;

(iv) a polypeptide having one or more conservative and/orsemi-conservative amino acid substitutions in SEQ ID NO: 46 or at least85%, 90%, 95%, 97%, 98%, or 99% sequence identity across the entirelength of SEQ ID NO: 46 and a D917A, E1006A, E1028A, D1255A, and/orN1257A amino acid substitution; or

(v) a polypeptide having one or more conservative and/orsemi-conservative amino acid substitutions in SEQ ID NO: 47 or at least85%, 90%, 95%, 97%, 98%, or 99% sequence identity across the entirelength of SEQ ID NO: 47 and a D901A and/or E1228A amino acidsubstitution.

86. The method of embodiment 83, wherein the guide RNA comprises asequence that is complementary to either strand of an ODP2 promotercomprising SEQ ID NO: 3 or SEQ ID NO: 71 or to either strand of an WUS2promoter comprising SEQ ID NO: 4, wherein the complementary sequence isimmediately adjacent to a protospacer adjacent motif (PAM) in SEQ ID NO:3, 71, or 4.

87. The method of embodiment 86, wherein the guide RNA comprises an RNAencoded by SEQ ID NO: 31, 32, 33, 34, or 35 that is complementary to asequence in the endogenous maize ODP2 promoter.

88. The method of embodiment 86, wherein the plant cell is a maize plantcell and the guide RNA comprises an RNA encoded by SEQ ID NO: 36, 37,38, 39, or 40 that is complementary to a sequence in the endogenousmaize WUS2 promoter.

89. The method of any one of embodiments 41 to 88, wherein at least twoexogenous gene transcription agents that stimulate transcription of theendogenous ODP2 gene and/or at least two exogenous gene transcriptionagents that stimulate transcription of the endogenous WUS2 gene areintroduced into a plant cell at step (i).

90. The method of embodiment 89, wherein the exogenous genetranscription agents comprise a transcriptional activation domain thatis operably linked to or operably associated with: (i) an artificialzinc finger (AZF) DNA binding domain polypeptide; (ii) an artificialTALE DNA binding polypeptide; (iii) an RNA guided DNA bindingpolypeptide that is nuclease activity deficient and a guide RNA, or anycombination thereof.

91. The method of embodiment 89, wherein the plant cell is a maize plantcell and the exogenous gene transcription agents that stimulatetranscription of the endogenous WUS2 gene comprise a combination of atleast two artificial TALE transcription factors comprising atranscriptional activation domain and an artificial TALE DNA bindingpolypeptide of SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:72,SEQ ID NO:75, or SEQ ID NO:78.

92. The method of embodiment 89, wherein the plant cell is a maize plantcell and the exogenous gene transcription agents that stimulatetranscription of the endogenous ODP2 gene comprise a combination of atleast two distinct artificial TALE transcription factors that eachcomprise a transcriptional activation domain that is operably linked toor operably associated with an artificial TALE DNA sequence recognitiondomain polypeptide of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:81, SEQ ID NO:84, or SEQ ID NO:87.

93. The plant cell of any one of embodiments 1, 5, 7, 8, 9, 15-20, or21, wherein the plant cell is a maize plant cell comprising at least oneexogenous gene transcription agent that stimulates transcription of theendogenous WUS2 gene, wherein expression of the endogenous WUS2polypeptide is increased in comparison to the expression of theendogenous WUS2 polypeptide in a control maize plant cell, wherein theendogenous WUS2 polypeptide is encoded by an endogenous polynucleotidethat is operably linked to an endogenous maize WUS2 promoter of SEQ IDNO:4 or an allelic variant thereof, wherein the exogenous genetranscription agent(s) bind to DNA sequences in the endogenous maizeWUS2 promoter corresponding to residues 100 to 225 of SEQ ID NO:4, andwherein the maize plant cell can form a regenerable maize plantstructure.

94. The maize plant cell of embodiment 93, wherein the exogenous genetranscription agent(s) bind to DNA sequences in the endogenous maizeWUS2 promoter corresponding to residues 130 to 210 of SEQ ID NO:4

95. The maize plant cell of embodiment 93, wherein an exogenouspolynucleotide encoding a WUS2 polypeptide is absent before and/orduring the increase in expression of the endogenous WUS2 polypeptide.

96. The maize plant cell of embodiment 93, wherein the increase inexpression of the endogenous WUS2 polypeptide is sufficient to increaseproliferation, somatic embryogenesis, and/or regeneration capacity ofthe maize plant cell in comparison to the control plant cell.

97. The maize plant cell of embodiment 93, wherein the increase inexpression of the endogenous WUS2 polypeptide is sufficient to increasetransformation efficiency and/or endogenous gene editing efficiency ofthe plant cell in comparison to the control plant cell.

98. The maize plant cell of embodiment 93, wherein said cell is locatedwithin or obtained from a cultured plant tissue explant, an immatureembryo, a mature embryo, a leaf, and/or callus or optionally wherein theplant cell is located with or derived from the L1 or L2 layer of theimmature or mature embryo.

99. The maize plant cell of embodiment 93, wherein the endogenous WUS2polypeptide comprises an amino acid sequence having at least 95%, 96%,97%, or 99% amino acid sequence identity across the entire length of SEQID NO:2.

100. The maize plant cell of embodiment 93, wherein the exogenous genetranscription agent comprises:

(i) a DNA binding domain which binds to the endogenous WUS2 gene; (ii) atranscription activation domain, wherein the transcription activationdomain is operably linked or operably associated with the DNA bindingdomain; and optionally (iii) an operably linked nuclear localizationsignal (NLS).

101. The maize plant cell of embodiment 100, wherein the DNA bindingdomain that binds the WUS2 promoter comprises an artificial zinc finger(AZF) DNA binding domain polypeptide.

102. The maize plant cell of embodiment 101, at least one AZF DNAbinding domain polypeptide binds to any one of SEQ ID NO:101 or 102.

103. The maize plant cell of embodiment 101, wherein at least twoexogenous gene transcription agents comprising an AZF DNA binding domainpolypeptide are provided and wherein one of the AZF DNA binding domainpolypeptides binds to a DNA sequence comprising SEQ ID NO:101 and one ofthe AZF DNA binding domain polypeptides binds to a DNA sequencecomprising SEQ ID NO:102.

104. The maize plant cell of embodiment 102, wherein the AZF DNA bindingdomain polypeptide comprises a polypeptide having at least 95%, 98%,99%, or 100% sequence identity to SEQ ID NO:105 or SEQ ID NO:106.

105. The maize plant cell of embodiment 101, wherein at least twoexogenous gene transcription agents comprising an AZF DNA binding domainpolypeptide are provided and wherein one of the AZF DNA binding domainpolypeptides comprises a polypeptide having at least 95%, 98%, 99%, or100% sequence identity to SEQ ID NO:105 and one of the AZF DNA bindingdomain polypeptides comprises a polypeptide having at least 95%, 98%,99%, or 100% sequence identity to SEQ ID NO:106.

106. The maize plant cell of embodiment 101, wherein the exogenous genetranscription agent comprises a polypeptide having at least 90%, 95%,98%, 99%, or 100% sequence identity to SEQ ID NO: 93 or 95.

107. The maize plant cell of embodiment 101, wherein at least twoexogenous gene transcription agents are provided and wherein one of theexogenous gene transcription agents comprises a polypeptide having atleast 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO:93 and oneof the exogenous gene transcription agents comprises a polypeptidehaving at least 95%, 98%, 99%, or 100% sequence identity to SEQ IDNO:95.

108. The method of any one of embodiments 41, 43-60, or 61 for producinga regenerable plant structure, wherein the regenerable plant structureis a regenerable maize plant structure, comprising: (i) introducing intoa maize plant cell at least one exogenous gene transcription agent whichtransiently increases expression of an endogenous WUS2 polypeptide,wherein the expression is increased in comparison to the expression ofthe endogenous WUS2 polypeptide in a control maize plant cell, whereinthe endogenous WUS2 polypeptide is encoded by an endogenouspolynucleotide that is operably linked to an endogenous maize WUS2promoter of SEQ ID NO:4 or an allelic variant thereof, and wherein theexogenous gene transcription agent(s) bind to DNA sequences in theendogenous maize WUS2 promoter corresponding to residues 100 to 225 ofSEQ ID NO:4; and, (ii) culturing the maize plant cell to produce aregenerable maize plant structure.

109. The method of embodiment 108, wherein the exogenous genetranscription agent comprises: (i) an artificial zinc finger (AZF) DNAbinding domain which binds to of the endogenous WUS2 gene; (ii) atranscription activation domain, wherein the transcription activationdomain is operably linked or operably associated with the domain orcomplex; and optionally (iii) an operably linked nuclear localizationsignal (NLS).

110. The method of embodiment 109, wherein at least one AZF DNA bindingdomain polypeptide binds to any one of SEQ ID NO:101 or 102 or whereinone AZF DNA binding domain polypeptides binds to a DNA moleculecomprising SEQ ID NO:101 and one of the AZF DNA binding domainpolypeptides binds to a DNA molecule comprising SEQ ID NO:102.

111. The method of embodiment 109, wherein the AZF DNA binding domainpolypeptide comprises a polypeptide having at least 95%, 98%, 99%, or100% sequence identity to SEQ ID NO:105 or SEQ ID NO:106.

112. The method of embodiment 109, at least two exogenous genetranscription agents comprising an AZF DNA binding domain polypeptideare provided and wherein one of the AZF DNA binding domain polypeptidescomprises a polypeptide having at least 95%, 98%, 99%, or 100% sequenceidentity to SEQ ID NO:105 and one of the AZF DNA binding domainpolypeptides comprises a polypeptide having at least 95%, 98%, 99%, or100% sequence identity to SEQ ID NO:106.

113. The method of embodiment 109, wherein the exogenous genetranscription agent comprises a polypeptide having at least 90%, 95%,98%, 99%, or 100% sequence identity to SEQ ID NO: 93 or 95.

114. The method of embodiment 109, wherein at least two exogenous genetranscription agents are provided and wherein one of the exogenous genetranscription agents comprises a polypeptide having at least 95%, 98%,99%, or 100% sequence identity to SEQ ID NO:93 and one of the exogenousgene transcription agents comprises a polypeptide having at least 95%,98%, 99%, or 100% sequence identity to SEQ ID NO:95.

115. The method of embodiment 108, wherein the exogenous genetranscription agent(s) bind to DNA sequences in the endogenous maizeWUS2 promoter corresponding to residues 130 to 210 of SEQ ID NO:4.

EXAMPLES Example 1 Artificial Zinc Finger Transcription Factors (ATF)for Increasing Expression of the Endogenous Maize ODP2 and WUS2 Genes

This example provides artificial Zinc Finger Transcription Factors (ATF)for increasing expression of the endogenous maize ODP2 and WUS2 genes.

Two high quality target binding sites for an ATF (SEQ ID NO: 6 and 9)were identified in the maize ODP2 promoter region of approximately 500bp (SEQ ID NO:3) which is proximal to the ODP2 gene transcriptioninitiation site (e.g., mRNA cap site). A ZNF DNA binding domain (SEQ IDNO:5) and an ATF comprising that DNA binding domain (SEQ ID NO:7) weredesigned to bind the ZmODP2 promoter at SEQ ID NO:6. A ZNF DNA bindingdomain (SEQ ID NO:8) and an ATF comprising that DNA binding domain (SEQID NO:10) was designed to bind the ZmODP2 promoter at SEQ ID NO:9. Eachof the ATFs comprise the maize opaque-2 nuclear localization signal, theartificial zinc finger DNA binding domain, and 60 amino acids from themaize C1 transcriptional activation domain.

Two high quality target binding sites for an ATF (SEQ ID NO:12 and 15)were identified in the maize WUS2 promoter region of approximately 500bp (SEQ ID NO:4) which is proximal to the WUS2 gene transcriptioninitiation site (e.g., mRNA cap site). A ZNF DNA binding domain (SEQ IDNO:11) and an ATF comprising that DNA binding domain (SEQ ID NO:13) wasdesigned to bind the ZmWUS2 promoter at SEQ ID NO:12. A ZNF DNA bindingdomain (SEQ ID NO:14) and an ATF comprising that DNA binding domain (SEQID NO:16) was designed to bind the ZmWUS2 promoter at SEQ ID NO:15. Eachof the ATFs comprise the maize opaque-2 nuclear localization signal, theartificial zinc finger DNA binding domain, and 60 amino acids from themaize C1 transcriptional activation domain.

Example 2 Artificial Transcription Activator-Like Effectors (aTALEs) forIncreasing Expression of the Endogenous Maize ODP2 and WUS2 Genes

This example provides artificial Transcription activator-like effectors(aTALEs) for increasing expression of the endogenous maize ODP2 and WUS2genes.

Three high quality target binding sites (SEQ ID NO: 65, 66, and 67) forthree aTALEs that are spaced at intervals of about 100 nucleotides wereidentified in the maize WUS2 promoter region of approximately 500 bp(SEQ ID NO:4) which is proximal to the WUS2 gene transcriptioninitiation site. A TALE DNA binding protein (SEQ ID NO:17) and an ATFcomprising that DNA binding protein (SEQ ID NO:18) was designed to bindthe ZmWUS2 promoter at SEQ ID NO:65. A TALE DNA binding protein (SEQ IDNO:19) and an ATF comprising that DNA binding protein (SEQ ID NO:20) wasdesigned to bind the ZmWUS2 promoter at SEQ ID NO:66. A TALE DNA bindingprotein (SEQ ID NO:21) and an ATF comprising that DNA binding protein(SEQ ID NO:22) was designed to bind the ZmWUS2 promoter at SEQ ID NO:67.Each of the ATFs comprise the DNA binding protein, SV40 NLS, a 3×FLAGsequence, a VP64 transcription activation domain, and a 6×His(Histidine) domain. The aTALE ATFs targeting the WUS2 promoter canfunction independently or in tandem.

Three high quality target binding sites (SEQ ID NO: 74, 77, and 80) forthree aTALEs were identified in the maize WUS2 promoter region ofapproximately 500 bp (SEQ ID NO:4) which is proximal to the WUS2 genetranscription initiation site. A TALE DNA binding protein (SEQ ID NO:72) and an ATF comprising that DNA binding protein (SEQ ID NO: 73) wasdesigned to bind the ZmWUS2 promoter at SEQ ID NO: 74. A TALE DNAbinding protein (SEQ ID NO: 75) and an ATF comprising that DNA bindingprotein (SEQ ID NO: 76) was designed to bind the ZmWUS2 promoter at SEQID NO: 77. A TALE DNA binding protein (SEQ ID NO:78) and an ATFcomprising that DNA binding protein (SEQ ID NO:79) was designed to bindthe ZmWUS2 promoter at SEQ ID NO: 80. Each of the ATFs comprise a 6×His(Histidine) domain, the DNA binding protein, an SV40 NLS, and a VP64transcription activation domain. The aTALE ATFs targeting the WUS2promoter can function independently or in tandem.

Three high quality target binding sites (SEQ ID NO: 68, 69, and 70) forthree aTALEs that are spaced at intervals of about 100 nucleotides wereidentified in the maize ODP2 promoter region of approximately 500 bp(SEQ ID NO: 3) which is proximal to the ODP2 gene transcriptioninitiation codon. A TALE DNA binding protein (SEQ ID NO:23) and an ATFcomprising that DNA binding protein (SEQ ID NO:24) was designed to bindthe ZmODP2 promoter at SEQ ID NO:68. A TALE DNA binding protein (SEQ IDNO:25) and an ATF comprising that DNA binding protein (SEQ ID NO:26) wasdesigned to bind the ZmODP2 promoter at SEQ ID NO:69. A TALE DNA bindingprotein (SEQ ID NO:27) and an ATF comprising that DNA binding protein(SEQ ID NO:28) was designed to bind the ZmODP2 promoter at SEQ ID NO:70.Each of the ATFs comprise the DNA binding domain, SV40 NLS, a 3×FLAGsequence, a VP64 transcription activation domain, and a 6×His(Histidine) domain. The three aTALE ATFs targeting the ODP2 promoter canfunction independently or in tandem.

Three high quality target binding sites (SEQ ID NO: 83, 86, and 89) forthree aTALEs were identified in the maize ODP2 promoter region ofapproximately 500 bp (SEQ ID NO: 3) which is proximal to the ODP2 genetranscription initiation codon. A TALE DNA binding protein (SEQ ID NO:81) and an ATF comprising that DNA binding protein (SEQ ID NO:82) wasdesigned to bind the ZmODP2 promoter at SEQ ID NO: 83. A TALE DNAbinding protein (SEQ ID NO: 84) and an ATF comprising that DNA bindingprotein (SEQ ID NO: 85) was designed to bind the ZmODP2 promoter at SEQID NO: 86. A TALE DNA binding protein (SEQ ID NO: 87) and an ATFcomprising that DNA binding protein (SEQ ID NO: 88) was designed to bindthe ZmODP2 promoter at SEQ ID NO: 89. Each of the ATFs comprise a 6×His(Histidine) domain, the DNA binding domain, SV40 NLS, and a VP64transcription activation domain. The three aTALE ATFs targeting the ODP2promoter can function independently or in tandem.

Example 3 Construction of dCas9 Transcription Activators and Guide RNAsTargeting ZmWUS2 and ZmODP2

This example describes an ATF comprising a nuclease deficient Cas9 DNAbinding domain, guide RNAs, and vectors useful for the expression of theguide RNAs. Such ATFs and guide RNAs are designed to increase expressionof the endogenous maize ODP2 and WUS2 genes.

Five crRNA (SEQ ID NO:31, 32, 33, 34, and 35) were constructed which arecomplementary to sequences immediately adjacent to PAM sequences in the−250 to −100 region of the maize ODP2 promoter (SEQ ID NO:3) relative tothe transcription start site. Five single guide RNAs (sgRNA)incorporating the crRNA can be obtained with the sgRNA expressioncassette of SEQ ID NO:41. The aforementioned sgRNAs can be expressed bysubstituting the aforementioned crRNA sequences for the 20 “N” (i.e.,a,c,g, or t) residues in the vector of SEQ ID NO: 41 which provides foroperable linkage of the crRNA sequences to a U6 promoter and sgRNAencoding sequences and introducing the vector with the substitution intoa suitable host cell (e.g., a meristematic cell, a somatic cell or areproductive cell). A dCas9 nuclease deficient RNA guided DNA bindingdomain (SEQ ID NO:29) is obtained and a polypeptide comprising that DNAbinding domain, an SV40 NLS, a 3×FLAG sequence, a VP64 transcriptionactivation domain, and a 6×His (Histidine) domain (SEQ ID NO:30) isdesigned to bind the ZmODP2 promoter when complexed with theaforementioned sgRNAs.

Five crRNA (SEQ ID NO: 36, 37, 38, 39 and 40) are constructed which arecomplementary to sequences immediately adjacent to PAM sequences in the−250 to −100 region of the maize WUS2 promoter (SEQ ID NO:4) relative tothe transcription start site. Five single guide RNAs (sgRNA)incorporating the crRNA can be obtained with the sgRNA expressioncassette of SEQ ID NO:41. The aforementioned sgRNAs can be expressed bysubstituting the aforementioned crRNA sequences for the 20 “N” (i.e.,a,c,g, or t) residues in the vector of SEQ ID NO: 41 which provides foroperable linkage of the crRNA sequences to a U6 promoter and sgRNAencoding sequences and introducing the vector with the substitution intoa suitable host cell (e.g., a meristematic cell, a somatic cell or areproductive cell). A dCas9 nuclease deficient RNA guided DNA bindingdomain (SEQ ID NO:29) is obtained and a polypeptide comprising that DNAbinding domain, an SV40 NLS, a 3×FLAG sequence, a VP64 transcriptionactivation domain, and a 6×His (Histidine) domain (SEQ ID NO:30) wasdesigned to bind the ZmWUS2 promoter when complexed with theaforementioned sgRNAs.

In certain cases, the dCas9 polypeptide is expressed in E. coli,purified, and complexed in vitro with the corresponding sgRNA fordelivery to a plant cell. Alternatively, the Cas9 protein and sgRNAs canbe expressed from a plasmid that is delivered to the target plant cellsor plant tissues.

Example 4 Use of ATFs to Obtain Regenerable Plant Structures

Developing maize embryos 8-14 days after pollination (DAP) from avariety or of a genotype that typically does not respond to biolistic oragrobacterium-mediated transformation are excised and placed on sterileplant growth media, scutellar side up. A plasmid encoding transcriptionactivation ATFs that target the maize ODP2 and WUS2 promoter proximalregions is delivered using biolistics. The plasmid may also encode amarker gene such as GFP (or variants thereof) or mCherry fluorescentproteins to identify cells containing the plasmid.

The expected positive result is formation of regenerable plantstructures comprising callus or pro-embryogenic masses from tissue thatreceived the plasmid containing the transcription activation ATF genesafter one week and no such formations on control tissue that receivedplasmid lacking the transcription activation ATF genes.

Example 5 Isolation of Corn Transformation Target Tissue and BiolisticDelivery of Transcription Activators in Target Tissue

Ears representing the target plant genotype are harvested approximately8-14 DAP. The tips of developing kernels are removed with a scalpel. Afine spatula is used to gently remove the embryo from the kernel, whichis then placed on callus induction media (e.g. 4 g L⁻¹ N6 salts plus N6vitamins, 2 mg L⁻¹ 2,4-D, 2.8 g L-proline, 30 g L⁻¹ sucrose, 100 mg L⁻¹casein hydrolysate, 100 mg L⁻¹ myo-inositol, 25 μM silver nitrate, 2.5 gL⁻¹ gelrite, pH 5.8). Embryo size is about 1.5-2.5 mm. Afterapproximately 200 embryos are harvested, they are arranged onto fourplates containing about 50 embryos each. Ideally, the scutellar surfaceis facing up.

The ATFs targeting ZmODP2 and ZmWUS2 (e.g., Zf ATF including SEQ IDNO:7, 10, 13, and/or 16; aTALE including SEQ ID NO:18, 20, 22, 24, 26,and/or 28; a dCas-NLS-TAD and guide RNAs including dCas9-NLS-TAD (SEQ IDNO: 30) and sgRNAs comprising crRNAs of SEQ ID NO:31, 32, 33, 34, 35,36, 37, 38, 39, and/or 40 are encoded on plasmid DNA using suitable genecassettes to drive their expression. The plasmid DNA is a standardhigh-copy E. coli vector that may or may not contain a selectable markergene. The plasmid DNA is prepared using standard molecular biologyprocedures, examined for integrity and quantified. The plasmid DNA iscomplexed with 0.6 μm gold particles as described, for example in(Hamada et al. 2018; K. Wang and Frame 2009). The embryos aretransferred to osmotic media (4 g L⁻¹ N6 salts plus N6 vitamins, 2 mgL⁻¹ 2,4-D, 0.7 g L-proline, 30 g L⁻¹ sucrose, 100 mg L⁻¹ caseinhydrolysate, 100 mg L⁻¹ myo-inositol, 36.4 g sorbitol, 36.4 g mannitol,25 μM silver nitrate, 2.5 g L⁻¹ gelrite, pH 5.8) four hours prior tobombardment. The gold particles are loaded into a BioRad PDS-1000 heliumgene gun and delivered to target plant tissue following manufacturer'sinstructions or variations developed by other researchers. Bombardedembryos are incubated overnight in the dark at 28° C.

The following morning embryos are moved onto callus induction media asabove, but with 0.8 mg L⁻¹ 2,4-D and cultured in dark at 28° C. forabout 5-7 days. The tissue is examined for the formation of regenerableplant structures comprising pro-embryogenic masses starting at 5 daysafter bombardment. ATF efficacy is scored on the basis ofpro-embryogenic mass formation compared to control tissue which receivedplasmid DNA lacking the ATF genes.

Example 6 Regeneration of Plants from Transformed Target Tissue

After 6-7 days on callus induction medium, the embryos are moved ontoshoot formation medium (e.g. MS, 60 g L⁻¹ sucrose, 0.5 mg L⁻¹ zeatin,0.1 mg L⁻¹ thidiazuron, 1 mg/L BAP, 0.1 mg L⁻¹ imazapyr, pH 5.8). After2 weeks on shoot formation medium, embryos are moved to rooting medium(e.g. MS, 40 g L⁻¹ sucrose, 0.1 mg L⁻¹ imazapyr, pH 5.8) and placedunder GE Ecolux® (General Electric; Boston, Mass.) fluorescent lights G(60 μmol m⁻² s⁻¹) with a 16-h photoperiod at 26° C. Once adequate shootswith roots form, plantlets are transferred to soil and grown to maturityin an appropriate growth environment like a greenhouse or growthchamber.

Example 7 Selection of Promoter Sequences Common Across Corn Lines forTargeting by ATFs

The promoter regions of ODP2 and WUS are sequenced and analyzed for thepresence of conserved regions. These consistent sequences are desirablebecause the same ATF reagents can be used in diverse corn germplasm toobtain regenerable plant structures. ATF reagents can be designed tobind and activate a conserved ODP2 promoter sequence of 312 bp (SEQ IDNO:71), that is 99% identical among B73, B104, PH207, Mo17, 2FACC,LH214, LH123HT and ICI441.

Example 8 Expression of Endogenous WUS2 in Corn Protoplasts Transfectedwith ATFs

Corn protoplasts were transfected with vectors expressing the ATFsZnFng-WUS1, 2, 3, and/or −4 (SEQ ID NOs: 93, 95, 97, and/or 99,respectively) or expressing GFP as a control. Endogenous RNA wasextracted, and the level of WUS2 mRNA expression is quantified by RT-PCRrelative to expression of the Act1 gene.

GFP, ZnFng-WUS3, or ZnFng-WUS4 expression did not significantly affectendogenous WUS2 transcription over background levels in mock transfectedprotoplasts. In contrast, ZnFng-WUS1 and ZnFng-WUS2 increased WUS2transcription over the background levels in mock transfectedprotoplasts. ZnFng-WUS1 promotes WUS2 expression increase to about 10%relative to actin and normalized by transfection efficiency (in the8-12% range in different experiments). ZnFng-WUS2 promoted a WUS2expression increase of about 50% relative to actin and normalized bytransfection efficiency (in the 15 to 60% range in differentexperiments). Mock transfected protoplasts had a nearly zero WUS2expression level relative to actin.

Example 9 Biological Sequences

This example provides non-limiting embodiments of proteins, promoters,and coding sequences referred to herein. Biological sequences and theirSEQ ID NOs are set forth in Table 1.

TABLE 1 Biological Sequences SEQ ID NO: DESCRIPTION SEQUENCE COMMENTS 1Endogenous MATVNNWLAFSLSPQELPPSQTTDS Maize TLISAATADHVSGDVCFNIPQDWSMODP2 RGSELSALVAEPKLEDFLGGISFSE polypeptide QHHKSNCNLIPSTSSTVCYASSAASTGYHHQLYQPTSSALHFADSVMVAS SAGVHDGGSMLSAAAANGVAGAASANGGGIGLSMIKNWLRSQPAPMQPRA AAAEGAQGLSLSMNMAGTTQGAAGMPLLAGERARAPESVSTSAQGGAVVV TAPKEDSGGSGVAGALVAVSTDTGGSGGASADNTARKTVDTFGQRTSIYR GVTRHRWTGRYEAHLWDNSCRREGQTRKGRQVYLGGYDKEEKAARAYDLA ALKYWGATTTTNFPVSNYEKELEDMKHMTRQEFVASLRRKSSGFSRGASI YRGVTRHHQHGRWQARIGRVAGNKDLYLGTFSTQEEAAEAYDIAAIKFRG LNAVTNFDMSRYDVKSILDSSALPIGSAAKRLKEAEAAASAQHHHAGVVS YDVGRIASQLGDGGALAAAYGAHYHGAAWPTIAFQPGAATTGLYHPYAQQ PMRGGGWCKQEQDHAVIAAAHSLQDLHHLNLGAAGAHDFFSAGQQAAAAA AMHGLASIDSASLEHSTGSNSVVYNGGVGDSNGASAVGSGGGYMMPMSAA GATTTSAMVSHEQMHARAYDEAKQAAQMGYESYLVNAENNGGGRMSAWGT VVSAAAAAAASSNDNIAADVGHGGA QLFSVWNDT 2Endogenous MAANAGGGGAGGGSGSGSVAAPAVC Maize RPSGSRWTPTPEQIRMLKELYYGCGWUS2 IRSPSSEQIQRITAMLRQHGKIEGK polypeptide NVFYWFQNHKARERQKRRLTSLDVNVPAAGAADATTSQLGVLSLSSPPSG AAPPSPTLGFYAAGNGGGSAGLLDTSSDWGSSGAAMATETCFLQDYMGVT DTGSSSQWPCFSSSDTIMAAAAAAARVATTRAPETLPLFPTCGDDDDDDS QPPPRPRHAVPVPAGETIRGGGGSSSSYLPFWGAGAASTTAGATSSVAIQ QQHQLQEQYSFYSNSTQLAGTGSQDVSASAAALELSLSSWCSPYPAAGSM 3 Endogenous AAATGGCCGTGACAACGTATACTAT500 bp promoter Maize TATCGAGTAAAAGGTCGCCACTTTA sequence; TATA ODP2GTAGTACATGTACATGCATGCGCAG box underlined; promoterATACATCATCAGGTACTCATATATG 5′-UTR in lower and GGCACACATATAGACATGTTTTGAGcase italics 5′-UTR GAAAATGAGACAAAGTATAGTGGAG ACTTCCCTAGAAAGCAGAAGAAAAAGAAGTGGTTTATGTTCCGTTAAATC ATACTACAACTTTTTTTTATTATACTCTCCATTTTGTCATCATTAGGTAC TCATATATGGGCACACATATAGTACTGCCAATTTTTCTTGCTAAAAAAAG TTCCACTATATATATGTATGTATGCACAAATAAACTAATTTTCTTAGAAA AGAAAACCGGTGTAATACATACTAAGGGCTAGTTTGGGAACCCTGGTTTT CTAAGGAATTTTATTTTTCCAAAAAAAATAGTTTATTTTTCCTTCGGAAA TTAGGAATCTCTTATA AAATTCGAGTTCCCAAACTATTCCTAATATATATatcatactctccat cagtctatatatagattacatatagtaagtatagagtatctcgctatcac atagtgccactaatcttctggagtgtaccagttgtataaatatctatcag tatcagcactactgtttgctgaataccccaaaactctctgcttgacttct cttccctaacctttgcactgtccaaaatggcttcctgatcccctcacttc ctcgaatcattctaagaagaaactcaagccgctaccattaggggcagatt aattgctgcactttcagataatcta cc 4 EndogenousCATTGAACAATGGAGCTGCAAGAGCAATGATG 609 bp WUS2 MaizeCACTAGCTAGTGTAATGCAGTGCATGCATGGT promoter and WUS2AGATTGGTAGCTAGCCTTTGCAGTTTGCACCA 5′UTR sequence: promoterGGCACCAGCAGCAGCTAGAAGACGACAGACG TATA box and ACAGGGGCTT

CAG underlined; 5′-UTR TTGCCAGTTGCCACAAGGGGAGCCTG

binding sites for

ATGATAGCTCTGTCTCTCTCA ZnFng-WUS1 CACACACACACAGTCACACAGAGACACGCAAA AZFDNATGACTTCTGTCTCTAACTCTTCCAAATTTCGAA BINDING SITEGCGGCCAATGCAAGAGCCAGCCCCCGGCCGT (SEQ ID NO:ATGTCAACTTCACTTGTCTCTCTCCAAAAGATA 101) atTCGTATCACCCATGGGCAATGGCCATGACCCC nucleotides 138-CCTCCCAGCCCCAACCTATATCACCTAGCGCAG 155 and ZnFng-CTACGCTCTCTTCTCCCGCTCTCGCTCTCTGCTG WUS2 AZF DNAGCTGCATGCTAGCTACCTTCTAGCTATCTAGCC BINDING SITETCTAGCTCCAATGCACTCCCTCCTTATAAACAA (ReverseGGAACCCTCCTTCGGCTCTCTTGCCATAGACCG complement ofGACACCGGAGAGCTAGGTCACAGAAGCGCTC SEQ ID NO: 102)AGGAAGGCCGCTGCGCTGAGATAGAGGC at nucleotides 185-202 to doubleunderlined and in bold 5 Zinc Finger DNA LEPGEKPYKCPECGKSFSQSSSLVRHQRTGEKP linkers binding domain THTGEKPYKCPECGKSFSRADNLTEHQ separating zinctargeting ZmODP2 RTHTGEKPYKCPECGKSFSTSGNLTEHQ fingers aregene sequence at RTHTGEKPYKCPECGKSFSTSGNLTEHQ underlined position 73RTHTGEKPYKCPECGKSFSQKSSLIAHQ RTHTGEKPYKCPECGKSFSRADNLTEH QRTHTGKKTS 6ZmODP2 gene 5′-cagatacatcatcaggta-3′ sequence at position 73 7Complete Zf ATF MrkrkesnresarrsrrsrvrkkvLEPGEKPYKCPE maize opaque-2targeting ZmODP2 CGKSFSQSSSLVRHQRTHTGEKPYKCPE nuclear at position 73CGKSFSRADNLTEHQRTHTGEKPYKCP localization signalECGKSFSTSGNLTEHQRTHTGEKPYKCP is lower case andECGKSFSTSGNLTEHQRTHTGEKPYKCP underlined; maizeECGKSFSQKSSLIAHQRTHTGEKPYKCP C1 transcriptionECGKSFSRADNLTEHQRTHTGKKTSAG activation domainSSDDCSSAASVSLRVGSHDEPCFSGDGD double underlinedGDWMDDVRALASFLESDEDWIRCQTA GQLA 8 Zinc Finger DNALEPGEKPYKCPECGKSFSQSGDLRRHQ binding domain RTHTGEKPYKCPECGKSFSTSGNLTEHQtargeting ZmODP2 RTHTGEKPYKCPECGKSFSQSSSLVRHQ gene sequence atRTHTGEKPYKCPECGKSFSTSGNLTEHQ position 51 RTHTGEKPYKCPECGKSFSQSSSLVRHQRTHTGEKPYKCPECGKSFSQSSSLVRHQ RTHTGKKTS 9 ZniODP2 gene5′-gtagtacatgtacatgca-3′ sequence at position 51 10 Complete Zf ATFMrkrkesnresarrsrrsrvrkkv maize opaque-2 targeting ZmODP2 LEPGEKPYKCPEnuclear at position 51 CGKSFSQSGDLRRHQRTHTGEKPYKCP localization signalECGKSFSTSGNLTEHQRTHTGEKPYKCP is lower case andECGKSFSQSSSLVRHQRTHTGEKPYKCP underlined; maizeECGKSFSTSGNLTEHQRTHTGEKPYKCP C1 transcriptionECGKSFSQSSSLVRHQRTHTGEKPYKCP ECGKSFSQSSSLVRHQRTHTGKKTSAGSactivation domain SDDCSSAASVSLRVGSHDEPCFSGDGD double underlinedGDWMDDVRALASFLFSDFDWLRCQTA GQLA 11 Zinc Finger DNALEPGEKPYKCPECGKSFSQAGHLASHQ binding domain RTHTGEKPYKCPECGKSFSTSGNLTEHQtargeting RTHTGEKPYKCPECGKSFSRSDKLTEHQ ZmWUS2 geneRTHTGEKPYKCPECGKSFSRNDALTEH sequence at QRTHTGEKPYKCPECGKSFSQSSNLVRposition 83 HQRTHTGEKPYKCPECGKSFSTTGNLT VHQRTHTGKKTS 12 ZmWUS2 gene5′-AATGAACTGCGGCATTGA-3′ sequence at position 83 13 Complete Zf ATFMrkrkesnresarrsrrsrvrkkvLEPGEKPYKCPE maize opaque-2 targetingCGKSFSQAGHLASHQRTHTGEKPYKCP nuclear ZmWUS2 atECGKSFSTSGNLTEHQRTHTGEKPYKCP localization signal position 83ECGKSFSRSDKLTEHQRTHTGEKPYKCP is lower case andECGKSFSRNDALTEHQRTHTGEKPYKC underlined; maizePECGKSFSQSSNLVRHQRTHTGEKPYK C1 transcription CPECGKSFSTTGNLTVHQRTHTGKKTSactivation domain AGSSDDCSSAASVSLRVGSHDEPCFSGD double underlinedGDGDWMDDVRALASFLFSDEDWLRCQ TAGQLA 14 Zinc Finger DNALEPGEKPYKCPECGKSFSRSDNLVRHQ binding domain RTHTGEKPYKCPECGKSFSRRDELNVHtargeting QRTHTGEKPYKCPECGKSFSSPADLTRH ZmWUS2 geneQRTHTGEKPYKCPECGKSFSQAGHLAS sequence at HQRTHTGEKPYKCPECGKSFSTSGNLTEposition 92 HQRTHTGEKPYKCPECGKSFSRSDKLTE HQRTHTGKKTS 15 ZmWUS2 gene5′-CGGCATTGAACAATGGAG-3′ sequence at position 92 16 Complete Zf ATFMrkrkesnresarrsrrsrvrkkvLEPGEKPYKCPE maize opaque-2 targetingCGKSFSRSDNLVRHQRTHTGEKPYKCP nuclear ZmWUS2 atECGKSFSRRDELNVHQRTHTGEKPYKC localization signal position 92PECGKSFSSPADLTRHQRTHTGEKPYKC is lower case andPECGKSFSQAGHLASHQRTHTGEKPYK underlined; maizeCPECGKSFSTSGNLTEHQRTHTGEKPYK C1 transcriptionCPECGKSFSRSDKLTEHQRTHTGKKTSA activation domainGSSDDCSSAASYSLRYGSHDEPCFSGDG double underlinedDGDWMDFWRALASFLESDEDWIRCQT AGQLA 17 ZmWUS2-TALE-Mapkkkrkvdykdhdgdykdhdidykdd N-terminus in 1 N-terminus,Ddkgtvdlrtlgysqqqqekikpkvrst lower case; RVD RVD, and C-Vaqhhealvghgfthahivalsqhpaal domain in in temiinal domainGtvavkyqdmiaalpeatheaivgvgkq uppercase with Wsgaralealltvagelrgpplqldtgqhypervariable Llkiakrggvtaveavhawrnaltgapl diresidues double Nltpeqvvaiunderlined; C- LTPEQVVAIASNHGGKQ terminus inALETVQRLLPVLCQAHGLTPEQVVAIAS lowercase italics;NHGGKQALETVQRLLPVLCQAHGLTPE Binds upstream ofQVVAIASHDGGKQALETVQRLLPVLCQ the ZmWUS2 AHGLTPEQVVAIASHDGGKQALETVQRtranscription start LLPVLCQAHGLTPEQVVAIASHDGGKQ siteALETVQRLLPVLCQAHGLTPEQVVAIAS NGGGKQALETVQRLLPVLCQAHLTPEQVVAIASHDGGKQALETVQRLLPVLCQA HGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNHGGKQA LETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQ VVAIASMGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRL LPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASH DGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQA HGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQA LETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQ VVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLL PVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASN HGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQA HGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQAL ETVQRLLPVLCQAHGLTPEQVVAIASNHGGKQALETVQRLLPVLCQAHGLTPEQ VVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGG sivaqlsrpdpalaaltndhlvalaclggrpaldavkkglphapalikrtnrripert shrva 18 ZmWUS2-TALE-mapkkkrkvdykdhdgdykdhdidy ZmWUS2-TALE- 1 Artificialkddddkgtvdlrtlgysqqqqekik 1 N terminus, Transcriptionpkvrstvaqhhealvghgfthahiv RVD domain, C- Factoralsqhpaalgtvavkyqdmiaalpe terminus in lower atheaivgvgkqwsgaralealltvcase; SV40 agelrgpplqldtgqllkiakrggv Nuclear taveavhawmaltgaplnltpeqvvLocalization ailtpeqvvaiasnhggkqaletvq Sequence inrllpvlcqahgltpeqvvaiasnhg uppercase, gkqaletvqrllpvlcqahgltpequnderlined; waiashdggkqaletvqrllpvlcq 3xFLAG ahgltpeqvvaiashdggkqaletvsequence qrllpvlcqahgltpeqvvaiashdg lowercase, gkqaletvqrllpvlcqahgltpequnderlined; VP64 vvaiasngggkqaletvqrllpvlc transcriptionalqahltpeqvvaiashdggkqaletv activation qrllpvlcqahgltpeqvvaiasngg domaingkqaletvqrllpvlcqahgltpeq is lowercase, vvaiasnhggkqaletvqrllpvlc doubleqahgltpeqvvaiashdggkqaletv underlined, qrllpvlcqahgltpeqvvaiasniitalics; ggkqaletvqrllpvlcqahgltpe 6xHis tag qvvaiasngggkqaletvqrllpvlat C-terminus cqahgltpeqvvaiashdggkqale tvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahglt peqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiashdggkqa letvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahg ltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiashdggk qaletvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqa hgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiasnhgg kqaletvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcq ahgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiasni ggkqaletvqrllpvlcqahgltpeqvvaiasnhggkqaletvqrllpvl cqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiasn gggsivaqlsrpdpalaaltndhlvalaclggrpaldavkkglphapali krtnrripertshrvaPKKKRKVSSdykdhdgdykdhdidykddddkAAG GGGSGRA

HHHHHH 19 ZmWUS2-TALE- Mapkkkrkvdykdhdgdykdhdidyk N-terminus in2 N-terminus, Ddddkgtvdlrtlgysqqqqekikpk lower case; RVD RVD, and C-vrstvaqhhealvghgfthahivals domain in in terminalqhpaalgtvavkyqdmiaalpeath uppercase with domaineaivgvgkqwsgaraleal1tvagel hypervariable rgpplqldtgqllkiakrggvtaveadiresidues vhawmaltgaplnltpeavvaiLTPE underlined; C- QVVAIASHDGGKQAterminus in LETVQRLLPVLCQAHGLTPEQVVAIAS lowercase italics;NGGGKQALETVQRLLPVLCQAHGLTPE Binds upstream ofQVVAIASNGGGKQALETVQRLLPVLCQ the ZmWUS2 AHGLTPEQVVAIASHDGGKQALETVQRtranscription LLPVLCQAHGLTPEQVVAIASHDGGKQ startALETVQRLLPVLCQAHGLTPEQVVAIAS site NIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQ ahgltpeqvvaiasniggkqaletvqrLLPVLCQAHGLTPEQVVAIASNGGGKQ ALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPE QVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQR LLPVLCQAHGLTPEQVVAIASNHGGKQALETVQRLLPVLCQAHGLTPEQVVAIAS NIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQ AHGLTPEQVVAIASNHGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQ ALETVQRLLPVLCQAHGLTPEQVVAIASNHGGKQALETVQRLLPVLCQAHGLTPE QVVAIASNHGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQR LLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIAS NIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQ AHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNHGGKQ ALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPE QVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQR LLPVLCQAHGLTPEQVVAIASNGGGsivaqlsrpdpalaaltndhlvalac lggrpaldavkkglphapalikrtnm pertshrva 20ZmWUS2-TALE- mapkkkrkvdykdhdgdykdhdidy ZmWUS2-TALE- 2 Artificialkddddkgtvdlrtlgysqqqqeki 2 N terminus, Transcriptionkpkvrstvaqhhealvghgfthahi RVD domain, C- Factorvalsqhpaalgtvavkyqdmiaalp terminus in lower eatheaivgvgkqwsgaralealltcase; SV40 vagelrgpplqldtgqllkiakrgg Nuclear vtaveavhawrnaltgaplnltpeqLocalization vvailtpeqvvaiashdggkqalet Sequence invqrllpvlcqahgltpeqvvaiasn uppercase, double gggkqaletvqrllpvlcqahgltpunderlined; eqvvaiasngggkqaletvqrllpv 3xFLAG lcqahgltpeqvvaiashdggkqalsequence etvqrllpvlcqahgltpeqvvaia lowercase, shdggkqaletvqrllpvlcqahglunderlined; VP64 tpeqvvaiasniggkqaletvqrll transcriptionalpvlcqahgltpeqvvaiasniggkq activation domain aletvqrllpvlcqahgltpeqvvais lowercase, iasniggkqaletvqrllpvlcqah double gltpeqvvaiasngggkqaletvqrunderlined, llpvlcqahgltpeqvvaiasngggk italics; 6xHis tagqaletvqrllpvlcqahgltpeqvv at C-terminus aiashdggkqaletvqrlIpvlcqahgltpeqvvaiashdggkqaletvq rllpvlcqahgltpeqvvaiasnhggkqaletvqrllpvlcqahgltpeqv vaiasniggkqaletvqrllpvlcqahgltpeqvvaiasniggkqaletv qrllpvlcqahgltpeqvvaiasnhggkqaletvqrlIpvlcqahgltpe qvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiasnhggkqale tvqrllpvlcqahgltpeqvvaiasnhggkqaletvqrllpvlcqahglt peqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiashdggkqa letvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahg ltpeqvvaiasniggkqaletvqrllpvlcqahgltpeqvvaiasngggk qaletvqrllpvlcqahgltpeqvvaiasnhggkqaletvqrllpvlcqa hgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiasnig gkqaletvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlc qahgltpeqvvaiasngggsivaqlsrpdpalaaltndhlvalaclggrp aldavkkglphapalikrtnrripe rtshrva

Sdykdhdgdy kdhdidykddddkAAGGGGSGRA

HHHHHH 21 Zm WUS2-T ALE- Mapkkkrkvdykdhdgdykdhdidyk N-terminus in3 N-termin ddddkgtvdlrtlgysqqqqekikpkv lower case; RVD us,rstvaqhhealvghgfthahivals domain in RVD, and C-qhpaalgtvavkyqdmiaalpeath uppercase with terminaleaivgvgkqwsgaralealltvage hypervariable domain lrgpplqldtgqllkiakrggvtavdi residues eavhawrnaltgaplnltpeqvvai underlined; C- LTPEQVVAIASNIGterminus in GKQALETVQRLLPVLCQAHGLTPEQ lowercaseVVAIASNGGGKQALETVQRLLPVLC italics; QAHGLTPEQVVAIASHD BindsGGKQALETVQRLLPVLCQAHGL upstream of TPEQVVAIASNHGG the ZmWUS2KQALETVQRLLPVLCQAHGLTPEQV transcription VAIASNGGGKQALETVQRLLPVLCQ startAHGLTPEQVVAIASNJGGKQALETV site QRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPE QVVAIASHDGGKQA LETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQ RLLPVLCQAHGLTPEQVVAIAS HDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHD GGKQALETVQRLLPVLCQAH GLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPE QVVAIASNIGGKQA LETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHG LTPEQVVAIASNHG GKQALETVQRLLPVLCQAHGLTPEQVVAIASNHGGKQAL ETVQRLLPVLCQAHGLTPEQVVAIA SHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIAS NIGGKQALETVQRLLPVL CQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIAS NHGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLC QAHGLTPEQVVAIASHD GGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGG KQALETVQRLLPVLCQAHGLTPEQV VAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIAS HDGGKQALETVQRLL PVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQ AHGLTPEQVVAIASHD GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGG sivaqlsrpdpalaaltndhlvala clggrpaldavkkglphapalikrtnrripertshrva 22 ZmWUS2-TALE- mapkkkrkvdykdhdgdykdhdidy ZmWUS2-TALE-3 Artificial kddddkgtvdlrtlgysqqqqekik 3 N terminus, Transcriptionpkvrstvaqhhealvghgfthahiv RVD domain, C- Factoralsqhpaalgtvavkyqdmiaalpe Terminus atheaivgvgkqwsgaralealltv in loweragelrgpplqldtgqllkiakrggv case; SV40 taveavhawrnaltgaplnltpeqv Nuclearvailtpeqvvaiasniggkqaletv Localization qrllpvlcqahgltpeqvvaiasngSequence in ggkqaletvqrllpvlcqahgltpe uppercase,qvvaiashdggkqaletvqrllpvl underlined; cqahgltpeqvvaiasnhggkqale 3xFLAGtvqrllpvlcqahgltpeqvvaias sequence ngggkqaletvqrllpvlcqahglt lowercase,peqvvaiasniggkqaletvqrllp underlined; vlcqahgltpeqvvaiasngggkqa VP64letvqrllpvlcqahgltpeqvvai transcriptional ashdggkqaletvqrllpvlcqahgactivation ltpeqvvaiasniggkqaletvqrl domain lpvlcqahgltpeqvvaiashdggkis lowercase, qaletvqrllpvlcqahgltpeqvv double aiashdggkqaletvqrllpvlcqahgltpeqvvaiashdggkqaletvq rllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpeq vvaiasngggkqaletvqrllpvlcqahgltpeqvvaiasnhggkqalet vqrllpvlcqahgltpeqvvaiasnhggkqaletvqrllpvlcqahgltp eqvvaiashdggkqaletvqrllpv lcqahgltpeqvvaiasniggkqaletvqrl underlined, lpvlcqahgltpeqvvaiasngggk italics;qaletvqrllpvlcqahgltpeqvv 6xHis tag aiasnhggkqaletvqrllpvlcqaat C-terminus hgltpeqvvaiasniggkqaletvq rllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeq vvaiashdggkqaletvqrllpvlcqahgltpeqvvaiashdggkqalet vqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltp eqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiashdggkqal etvqrllpvlcqahgltpeqvvaiasngggsivaqlsrpdpalaaltndh lvalaclggrpaldavkkglphapalikrtnrripertshrvaPKKKRKV SSdykdhdydykdhdidykddddkA AGGGGSGRA

HHHHHH 23 Zm ODP2-TALE- mapkkkrkvdykdhdgdykdhdidy N-terminus in1 N-terminus, kddddkgtvdlrtlgysqqqqekik lower case; RVD RVD, and C-pkvrstvaqhhealvghgfthahiv domain in terminal domainalsqhpaalgtvavkyqdmiaalpe uppercase with atheaivgvgkqwsgaralealltvhypervariable agelrgpplqldtgqllkiakrggv di residuestaveavhawrnaltgaplnltpeqv underlined; C- vaiLTPEQVVAIASNI terminus inGGKQALETVQRLLPVLCQAHGLT lowercase PEQVVAIASNGGGK italics;QALETVQRLLPVLCQAHGLTPEQVV Binds AIASNGGGKQALET upstream ofVQRLLPVLCQAHGLTPEQVVAIAS the ZmODP2 HDGGKQALETVQRLL transcriptionPVLCQAHGLTPEQVVAIAS start HDGGKQALETVQRLLPVLCQ site AHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLT PEQVVAIASNIGGK QALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALET VQRLLPVLCQAHGLTPEQVVAIAS NGGGKQALETVQRLLPVLCQAHGLTPEQVVAIA NI GGKQALETVQRLLPVLCQ AHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLT PEQVVAIASMGGKQALETVQRLLPV LCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAH GLTPEQVVAIASNI GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQA LETVQRLLPVLCQAHGLTPEQVVAI ASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIAS NGGGKQALETVQRLLPV LCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAH GLTPEQVVAIASNI GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQA LETVQRLLPV LCQAHGLTPEQVVAIAS NIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHD GGKQALETVQRLLPVLCQAHGLTPE QVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAI ASHDGGKQALETVQ RLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPV LCQAHGLTPEQVVAIASHD GGKQALETVQRLLPVLCQAHGLTPEQVVAIASHD GGKQALETVQRLLPVLCQAHGLTPE QVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAI ASNGGGsivaqlsv rpdpalaaltndhlvalaclggrpaldavkkglphapalikrfnrriper tshrva 24 Zm ODP2-TALE-mapkkkrkvdykdhdgdykdhdidyk Zm ODP2-TALE- 1 Artificialddddkgtvdlrtlgysqqqqekikp 1 N terminus, Transcriptionkvrstvaqhhealvghgfthahiva RVD domain, C- Factorlsqhpaalgtvavkyqdmiaalpea terminus theaivgvgkqwsgaralealltva in lowergelrgpplqldtgqllkiakrggvt case; SV40 aveavhawrnaltgaplnltpeqvv Nuclearailtpeqvvaiasniggkqaletvq Localization rllpvlcqahgltpeqvvaiasnggSequence in gkqaletvqrllpvlcqahgltpeq uppercase,vvaiasngggkqaletvqrllpvlc underlined; qahgltpeqvvaiashdggkqalet 3xFLAGvqrllpvlcqahgltpeqvvaiash sequence dggkqaletvqrllpvlcqahgltp lowercase,eqvvaiasngggkqaletvqrllpv underlined; lcqahgltpeqvvaiasniggkqal VP64etvqrllpvlcqahgltpeqvvaia transcriptional sniggkqaletvqrllpvlcqahglactivation tpeqvvaiasngggkqaletvqrll domain pvlcqahgltpeqvvaiasniggkqis lowercase, aletvqrllpvlcqahgltpeqvva double iasngggkqaletvqrllpvlcqahunderlined, gltpeqvvaiasniggkqaletvqr italics; llpvlcqahgltpeqvvaiasnggg6xHis tag kqaletvqrllpvlcqahgltpeqv at C-terminusvaiasniggkqaletvqrllpvlcq ahgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiasni ggkqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvl cqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaias niggkqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllp vlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpeqvvai ashdggkqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrl lpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvv aiasngggkqaletvqrllpvlcqahgltpeqvvaiashdggkqaletvq rllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeq vvaiasniggkqaletvqrllpvlcqahgltpeqvvaiasngggsivaql srpdpalaaltndhlvalaclggrpaldavkkglphapalikrtnrripe rtshrvaPKKKRKVSS dykdhdgdykdhdidykddddkAAGGGGSGRAd

HHHHHH 25 Zm ODP2-TALE- mapkkkrkvdykdhdgdykdhdidy N-terminus in2 N-terminus, kddddkgtvdlrtlgysqqqqekik lower case; RVD RVD, and C-pkvrstvaqhhealvghgfthahiv domain in terminal alsqhpaalgtvavkyqdmiaalpeuppercase with domain atheaivgvgkqwsgaralealltv hypervariableagelrgpplqldtgqllkiakrggv di residues taveavhawrnaltgaplnltpeqvunderlined; C- vaiLTPEQVVAIASHD terminus in GGKQALETVQRLLPVLCQAHGLTlowercase italics; PEQVVAIASNGGGK Binds upstream ofQALETVQRLLPVLCQAHGLTPEQVV the ZmODP2 AIASNGGGKQALET transcription startVQRLLPVLCQAHGLTPEQVVAIAS site HDGGKQALETVQRLL PVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQ AHGLTPEQVVAIASNH GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNHGGK QALETVQRLLPVLCQAHGLTPEQVV AIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIAS NHGGKQALETVQRLL PVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQ AHGLTPEQVVAIASNH GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGK QALETVQRLLPVLCQAHGLTPEQVV AIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIAS HDGGKQALETVQRLL PVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQ AHGLTPEQVVAIASMGGKQALETVQ RLLPVLCQAHGLTPEQVVAIASNHGGKQALETVQRLLPV LCQAHGLTPEQVVAIASNG GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNG GGKQALETVQRLLPVLCQAHGLTPE QVVAIASNHGGKQALETVQRLLPVLCQAHGLTPEQVVAI ASNGGGKQALETVQ RLLPVLCQAHGLTPEQVVAIASN1GGKQALETVQRLLPVLCQAHGLTPEQ VVAIASNGGGKQAL ETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQR LLpvlcqahgltpeqvvaiasmggk qaleTVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALET VQRLLPVLCQAHGLTPEQVVAIAS NGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNI GGKQALETVQRLLPVLCQAHGLTP EQVVAIASNGGGsivaqlsrpdpalaaltndhlv alaclggrpaldavkkglphap alikrtnrripertshrva 26Zm ODP2-TALE- mapkkkrkvdykdhdgdykdhdidykdd N terminus, RVD 2 Artificialddkgtvdlrtlgysqqqqekikpkvr domain, C- Transcriptionstvaqhhealvghgfthahivalsq terminus Factor hpaalgtvavkyqdmiaalpeathein lower aivgvgkqwsgaralealltvagel case; SV40 rgpplqldtgqllkiakrggvtaveNuclear avhawrnaltgaplnltpeqvvailt Localizationpeqvvaiashdggkqaletvqrllp Sequence in vlcqahgltpeqvvaiasngggkqauppercase, letvqrllpvlcqahgltpeqvvai underlined;asngggkqaletvqrllpvlcqahg 3xFLAG ltpeqvvaiashdggkqaletvqrl sequencelpvlcqahgltpeqvvaiasngggk lowercase, qaletvqrllpvlcqahgltpeqvvunderlined; aiasnhggkqaletvqrllpvlcqa VP64 hgltpeqvvaiasnhggkqaletvqtranscriptional rllpvlcqahgltpeqvvaiasnig activationgkqaletvqrllpvlcqahgltpeq domain vvaiasnhggkqaletvqrllpvlc is lowercase,qahgltpeqvvaiasngggkqalet double vqrllpvlcqahgltpeqvvaiasn underlined,hggkqaletvqrllpvlcqahgltp italics; eqvvaiasngggkqaletvqrllpv 6xHis taglcqahgltpeqvvaiasniggkqal at C-terminus etvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgl tpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiasniggkq aletvqrllpvlcqahgltpeqvvaiasnhggkqaletvqrllpvlcqah gltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiasngggk qaletvqrllpvlcqahgltpeqvvaiasnhggkqaletvqrllpvlcqa hgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiasnig gkqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvlc qahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpeqvvaiasn iggkqaletvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpv lcqahgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaia sniggkqaletvqrllpvlcqahgltpeqvvaiasngggsivaqlsrpdp alaaltndhlvalaclggrpaldavkkalgphaalikrtnrripertshrv a

SS dykdhdgdykdhdidykddddk AAGGGGSGRA

HHHHHH 27 Zm ODP2-TALE- mapkkkrkvdykdhdgdykdhdidy N-terminus in3 N-terminus, kddddkgtvdlrtlgysqqqqekik lower case; RVD RVD, and C-pkvrstvaqhhealvghgfthahiv domain in terminal alsqhpaalgtvavkyqdmiaalpeuppercase with domain atheaivgvgkqwsgaralealltv hypervariableagelrgpplqldtgqllkiakrggv diresidues taveavhawrnaltgaplnltpeqvunderlined; C- vaiLTPEQVVAIASNHG terminus in GKQALETVQRLLPVLCQAHGLTlowercase PEQVVAIASHDGGKQA italics; LETVQRLLPVLCQAHGLTPEQVV BindsAIASNIGGKQALETVQ upstream of RLLPVLCQAHGLTPEQVVAIAS the ZmODP2HDGGKQALETVQRLL transcription PVLCQAHGLTPEQVVAIASNG startGGKQALETVQRLLPVLCQ site AHGLTPEQVVAIASNH GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGK QALETVQRLLPVLCQAHGLTPEQVV AIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIAS HDGGKQALETVQRLL PVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQ AHGLTPEQVVAIASNI GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGK QALETVQRLLPVLCQAHGLTPEQVV AIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIAS NGGGKQALETVQRLL PVLCQAHGLTPEQVVAIASNHGGKQALETVQRLLPVLCQ AHGLTPEQVVAIASNH GGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGK QALETVQRLLPVLCQAHGLTPEQVV AIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIAS NGGGKQALETVQRLL PVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQ AHGLTPEQVVAIASHD GGKQALETVQRLLPVLCQAHGPLTEQVVAIASNGGGK QALETVQRLLPVLCQAHGLTPE QVVAIASNHGGKQALETVQRLLPVLCQAHGLTPEQVVAIASM GGKQALETVQRLLPVLCQAHGLTPE QVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAI ASHDGGKQALETVQ RLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPV LCQAHGLTPEQVVAIASH DGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHD GGKQALETVQRLLPVLCQAHGLTP EQVVAIASNGGGsivaqlsrpdpalaaltndhlv alaclggrpaldavkkglphap alikrtnrripertshrva 28Zm ODP2-TALE- mapkkkrkvdykdhdgdykdhdidy N terminus, RVD 3 Artificialkddddkgtvdlrtlgysqqqqekik domain, C- TranscriptiQnpkvrstvaqhhealvghgfthahiv terminus in lower FactQralsqhpaalgtvavkyqdmiaalpe case; SV40 atheaivgvgkqwsgaralealltv Nuclearagelrgpplqldtgqllkiakrggv Localization taveavhawrnaltgaplnltpeqvSequence in vailtpeqvvaiasnhggkqaletv uppercase,qrllpvlcqahgltpeqvvaiashd underlined; ggkqaletvqrllpvlcqahgltpe 3xFLAGqvvaiasniggkqaletvqrllpvl sequence cqahgltpeqvvaiashdggkqale lowercase,tvqrllpvlcqahgltpeqvvaias underlined; VP64 ngggkqaletvqrllpvlcqahglttranscriptional peqvvaiasnhggkqaletvqrllp activation domainvlcqahgltpeqvvaiasngggkqa is lowercase, letvqrllpvlcqahgltpeqvvai doubleashdggkqaletvqrllpvlcqahg underlined, ltpeqvvaiashdggkqaletvqrlitalics; 6xHis tag lpvlcqahgltpeqvvaiasniggk at C-terminusqaletvqrllpvlcqahgltpeqvv aiasniggkqaletvqrllpvlcqahgltpeqvvaiasniggkqaletvq rllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpeq vvaiasngggkqaletvqrllpvlcqahgltpeqvvaiasnhggkqalet vqrllpvlcqahgltpeqvvaiasnhggkqaletvqrllpvlcqahgltp eqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiasngggkqal etvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqahgl tpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiashdggkq aletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqah gltpeqvvaiasnhggkqaletvqrllpvlcqahgltpeqvvaiasnigg kqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcq ahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiashd ggkqaletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvl cqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaias ngggsivaqlsrpdpalaaltndhlvalaclggrpaldavkkglphapal ikrtnrripertshrva PKKKRKVSSdykdhdgdykdhdidykddddk AAGGGGSGRA

HHHHHH 29 dCAS9 RNA mdkkysiglaigtnsvgwavitdey dCas9 domain guided DNAkvpskkfkvlgntdrhsikknliga binding llfdsgetaeatrlkrtarrrytrr polypeptideknricylqeifsnemakvddsffhr leesflveedkkherhpifgnivdevayhekyptiyhlrkklvdstdkad lrliylalahmikfrghfliegdlnpdnsdvdklfiqlvqtynqlfeenp inasgvdakailsarlsksrrlenliaqlpgekknglfgnlialslgltp nfksnfdlaedaklqlskdtydddldnllaqigdqyadlflaaknlsdai llsdilrvnteitkaplsasmikrydehhqdltllkalvrqqlpekykei ffdqskngyagyidggasqeefykfikpilekmdgteellvklnredllr kqrtfdngsiphqihlgelhailrrqedfypflkdnrekiekiltfripy yvgplargnsrfawmtrkseetitpwnfeewdkgasaqsfiermtnfdkn lpnekvlpkhsllyeyftvyneltkvkyvtegmrkpaflsgeqkkaivdl lfktnrkvtvkqlkedyfkkiecfdsveisgvedrfnaslgtyhdllkii kdkdfldneenediledivltltlfedremieerlktyahlfddkvmkql krrrytgwgrlsrklingirdkqsgktildflksdgfanmfmqlihddsl tfkediqkaqvsgqgdslhehianlagspaikkgilqtvkwdelvkvmgr hkpeniviemarenqttqkgqknsrermkrieegikelgsqilkehpven tqlqneklylyylqngrdmyvdqeldinrlsdydvdaivpqsflkddsid nkvltrsdknrgksdnvpseevvkkmknywrqllnaklitqrkfdnltka ergglseldkagfikrqlvetrqitkhvaqildsrmntkydendklirev kvitlksklvsdfrkdfqfykvreinnyhhahdaylnavvgtalikkypk lesefvygdykvydvrkmiakseqeigkatakyffysnimnffkteitla ngeirkrpiietngetgeivwdkgrdfatvrkvlsmpqvnivkktevqtg gfskesilpkrnsdkliarkkdwdpkkyggfdsptvaysvlvvakvekgk skklksvkellgitimerssfeknpidfleakgykevkkdliiklpkysi felengrkrmlasagelqkgnelalpskyvnflylashyeklkgspedne qkqlfveqhkhyldeiieqisefskrviladanldkvlsaynkhrdkpir eqaeniihlftltnlgapaafkyfdttidrkrytstkevldatlihqsit glyetridlsqlggd 30 dCAS9 RNAmdkkysiglaigtnsvgwavitdey dCas9 domain is guided DNAkvpskkfkvlgntdrhsikknliga in lower case, bindingllfdsgetaeatrlkrtarrrytrr SV40 NLS is polypeptideknricylqeifsnemakvddsffhr uppercase and with leesflveedkkherhpifgnivdeunderlined, Nuclear vayhekyptiyhlrkklvdstdkad 3xFLAG Localizationlrliylalahmikfrghfliegdln sequence is lower andpdnsdvdklfiqlvqtynqlfeenp case and Transcriptioninasgvdakailsarlsksrrlenl underlined, the Activatoriaqlpgekknglfgnlialslgltp VP64 domain is sequencesnfksnfdlaedaklqlskdtydddl lower case, with C- dnllaqigdqyadlflaaknlsdaiunderlined, and terminal llsdilrvnteitkaplsasmikry italicized; 6xHis6xHis dehhqdltllkalvrqqlpekykei domain at C- domainffdqskngyagyidggasqeefykf terminus ikpilekmdgteellvklnredllrkqrtfdngsiphqihigelhailrr qedfypflkdnrekiekiltfripyyvgplargnsrfawmtrkseetitp wnfeevvdkgasaqsfiermtnfdknlpnekvlpkhsllyeyftvynelt kvkyvtegmrkpaflsgeqkkaivdllfktnrkvtvkqlkedyfkkiecf dsveisgvedrfnaslgtyhdllkiikdkdfldneenediledivltltl fedremieerlktyahlfddkvmkqlkrrrytgwgrlsrklingirdkqs gktildflksdgfanmfmqlihddsltfkediqkaqvsgqgdslhehian lagspaikkgilqtvkvvdelvkvmgrhkpeniviemarenqttqkgqkn srermkrieegikelgsqilkehpventqlqneklylyylqngrdmyvdq eldinrlsdydvdaivpqsflkddsidnkvltrsdknrgksdnvpseevv kkmknywrqllnaklitqrkfdnltkaergglseldkagfikrqlvetrq itkhvaqildsrmntkydendklirevkvitlksklvsdfrkdfqfykvr ei nnyhhahdaylnavvgtalikkypklesefvygdykvydvrkmiakseqei gkatakyffysnimnffkteitlangeirkrplietngetgeivwdkgrd fatvrkvlsmpqvnivkktevqtggfskesilpkrnsdkliarkkdwdpk kyggfdsptvaysvlvvakvekgkskklksvkellgitimerssfeknpi dfleakgykevkkdliiklpkysifelengrkrmlasagelqkgnelalp skyvnflylashyeklkgspedneqkqlfveqhkhyldeiieqisefskr viladanldkvlsaynkhrdkpireqaeniihlftltnlgapaafkyfdt tidrkrytstkevldatlihqsitg lyetridlsalggdGSPKKKRKVSS dykdhdgdykdhdidykddddk AAGGGGSGRAd

HHHHHH 31 ODP2_promoter_c ttatttttccttcggaaatt T (Thymine)rRNA-1 encoding residues are U sequence (uracil) residues in encoded RNA32 ODP2_promoter_c AAAATAGTTTATTTTTCCTT T (Thymine) rRNA-2 encodingresidues are U sequence (uracil) residues in encoded RNA 33ODP2_promoter_c TTGGGAACCCTGGTTTTCTA T (Thymine) rRNA-3 encodingresidues are U sequence (uracil) residues in encoded RNA 34ODP2_promoter_c AAGGGCTAGTTTGGGAACCC T (Thymine) rRNA-4 encodingresidues are U sequence (uracil) residues in encoded RNA 35ODP2_promoter_c TACATACTAAGGGCTAGTTT T (Thymine) rRNA-5 encodingresidues are U sequence encoding (uracil) residues sequencein encoded RNA 36 WU S2_promoter_ AAAAGATATCGTATCACCCA T (Thymine)crRNA-1 encoding residues are U sequence (uracil) residuesin encoded RNA 37 WUS2_promoter_ GCAATGCAAGAGCCAGCCCC T (Thymine)crRNA-2 encoding residues are U sequence (uracil) residuesin encoded RNA 38 WUS2_promoter_ GACTCTTCCAAATTCCGAAG T (Thymine)crRNA-3 residues are U encoding (uracil) residues sequencein encoded RNA 39 WUS2_promoter_ CAGTTGCCACAAGGGGAGCC T (Thymine)crRNA-4 residues are U encoding (uracil) residues sequencein encoded RNA 40 WUS2_promoter GGCAGTTACCAGTTGCCACA T (Thymine) crRNA-5residues are U encoding (uracil) residues sequence in encoded RNA 41sgRNA expression aaaataaatggtaaaatgtcaaatcaaaactaggctgcagtatGmU6 promoter cassette gcagagcagagtcatgatgatactacttactacaccgattcttgin lower case; for the tgtgcagaaaaatatgttaaaataattgaatctttctctagencoded sgRNA tethering ccaaatttgacaacaatgtacaccgttcatattgagagacgtarget sequence is approach atgcttcttgtttgctttcggtggaagctgcatatactcaain upper case, cattactccttcagcgagttttccaactgagtcccacatt wherein the Nx20gcccagacctaacacggtattcttgtttataatgaaatgt sequence can begccaccacatggattgNNNNNNNNNNNNNNNNNNNN SEQ ID NO: 31,gttttagagctagaaatagcaagttaaaataaggctagtccg 32, 33, 34, 35, 36,ttatcaacttgaaaaagtggcaccgagtcggtgctttttt 37, 38, 39, or 40; or any othercrRNA; remainder of the encoded sgRNA is in lower case and underlined 42maize opaque-2 RKRKESNRESARRSRRSRYRKKV nuclear localization signal 4360 amino acids AGSSDDCSSAASVSLRVGSHDEPCFSGD from theGDGDWMDDVRALASFLESDEDWLRCQ maize C1 TAGQLA transcriptional activationdomain 44 As Cpfl (wild MTQFEGFTNLYQVSKTLRFELIPQGKTL Acidaminococcus+Ltype) KHIQEQGFIEEDKARNDHYKELKPIIDRI sp. (As) dCpf1YKTYADQCLQLVQLDWENLSAAIDSYR variants includeKEKTEETRNALIEEQATYRNAIHDYFIG D917A, E1006A, RTDNLTDAINKRHAEIYKGLFKAELFNGE1028A, KVLKQLGTVTTTEFIENALLRSFDKFTT D1255A, and/orYFSGFYENRKNVFSAEDISTAIPHRIVQD N1257A amino NFPKFKENCHIFTRLITAVPSLREHFENVacid substitutions KKAIGIFVSTSIEEVFSFPFYNQLLTQTQI in the wild typeDLYNQLLGGISREAGTEKIKGLNEVLNL sequence; wild-AIQKNDETAHIIASLPHRFIPLFKQILSDR type residues thatNTLSFILEEFKSDEEVIQSFCKYKTLLRN can be substitutedENVLETAEALFNELNSIDLTHIFISHKKL to obtain dCpflETISSALCDHWDTLRNALYERRISELTG variants shown inKITKSAKEKVQRSLKHEDINLQEIISAAG bold and KELSEAFKQKTSEILSHAHAALDQPLPTunderlined TLKKQEEKEILKSQLDSLLGLYHLLDWF AVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPT LASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDK MYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPE KEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQY KDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPN LHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKD QKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFF FHVPITLNYQAANSPSKFNQRVNAYLK EHPETPIIGI DRGERNLIYITVIDSTGKILE QRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDL MIHYQAVVVL E NLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKV GGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNH ESRKHFLEGFDFLHYDVKTGDFILFLFKMNRNLSFQRGLPGFMPAWDIVFEKNET QFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPK LLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPE WPMDA D ANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRN 45 LbCpf1 (wild MSKLEKFTNCYSLSKTLRFKAIPVGKTQLachnospiraceae type) ENIDNKRLLVEDEKRAEDYKGVKKLLD bacterium (Lb)RYYLSFINDVLHSIKLKNLNNYISLFRK DCpf1 variants KTRTEKENKELENLEINLRKEIAKAFKGinclude D832A, NEGYKSLFKKDIIETILPEFLDDKDEIAL E925A, and/orVNSFNGFTTAFTGFFDNRENMFSEEAKS D1148A amino TSIAFRCINENLTRYISNMDIFEKVDAIFacid substitutions DKHEVQEIKEKILNSDYDVEDFFEGEFF in theNFVLTQEGIDVYNAIIGGFVTESGEKIKG wild type LNEYINLYNQKTKQKLPKFKPLYKQVLsequence; wild- SDRESLSFYGEGYTSDEEVLEVFRNTLN type residues thatKNSEIFSSIKKLEKLFKNFDEYSSAGIFV can be substitutedKNGPAISTISKDIFGEWNVIRDKWNAEY to obtain dCpf1 DDIHLKKKAVVTEKYEDDRRKSFKKIGvariants SFSLEQLQEYADADLSVVEKLKEIIIQKV shown inDEIYKVYGSSEKLFDADFVLEKSLKKN bold and DAVVAIMKDLLDSVKSFENYIKAFFGEunderlined GKETNRDESFYGDFVLAYDILLKVDHIY DAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAI MDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPS EDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYK DIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPN LHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELWHPANSPIANKNPDN PKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYV IGI D RGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFE ARQNWTSIENIKELKAGYISQVVHKICE LVEKYDAVIAL EDLNSGFKNSRVKVEK QVYQKFEKMLIDKLNYMVDKKSNPCA TGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIAD SKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPK KNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSL MLQMRNSITGRTDV D FLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIA RKVLWAIGQFKKAEDEKLDKVKIAISN KEWLEYAQTSVKH 46Fn Cpf1 (wild MSIYQEFVNKYSLSKTLRFELIPQGKTL Francisella type)ENIKARGLILDDEKRAKDYKKAKQIIDK novicida (Fn) YHQFFIEEILSSVCISEDLLQNYSDVYFKdCpfl variants LKKSDDDNLQKDFKSAKDTIKKQISEYI include FnCpf1KDSEKFKNLFNQNLIDAKKGQESDLIL amino acid WLKQSKDNGIELFKANSDITDIDEALEIIsubstitutions KSFKGWTTYFKGFHENRKNVYSSNDIP inTSIIYRIVDDNLPKFLENKAKYESLKDK the wild type APEAINYEQIKKDLAEELTFDIDYKTSEsequence; wild- VNQRVFSLDEVEEIANFNNYLNQSGITK type residues thatFNTIIGGKFVNGENTKRKGINEYINLYS can be substitutedQQINDKTLKKYKMSVLFKQILSDTESKS to obtain FVTDKLEDDSDVVTTMQSFYEQIAAFKTdCpf1 VEEKSIKETLSLLFDDLKAQKLDLSKIYF variantsKNDKSLTDLSQQVFDDYSVIGTAVLEYI shown in TQQIAPKNLDNPSKKEQELIAKKTEKAKbold and YLSLETIKLALEEFNKHRDIDKQCRFEEI underlinedLANFAAIPMIFDEIAQNKDNLAQISIKYQ NQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHF YLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDN TAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPK VFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSIS KHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLY LFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIP KKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGAN KFNDEINLLLKEKANDVHILSI D RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMK TNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF E DLNFGFKRGRFKVEKQVYQKL E KMLIEKLNYLWKDNEFDKTGGVLRAYQLTA PFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKI CYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREV YPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGT ELDYLISPVADVNGNFFDSRQAPKNMP QDA D A NGAYHIGLKGLMLLGRIKNNQ EGKKLNLVIKNEEYFEFVQNRNN 47 CasJ (wild type)MQQYQVSKTVRFGLTLKNSEKKHATH dCasJ variants LLLKDLVNVSEERIKNEITKDDKNQSELinclude D901A SFFNEVIETLDLMDKYIKDWENCFYRT and/or El228ADQIQLTKEYYKVIAKKACFDWFWTND amino acid RGMKFPTSSIISFNSLKSSDKSKTSDNLDsubstitutions in RKKKILDYWKGNIFKTQKAIKDVLDITE the wild typeDIQKAIEEKKSHREINRVNHRKMGIHLI sequence; wild-HLINDTLVPLCNGSIFFGNISKLDFCESE type residuesNEKLIDFASTEKQDERKFLLSKINEIKQY that can be FEDNGGNVPFARATLNRHTANQKPDRYsubstituted NEEIKKLVNELGVNSLVRSLKSKTIEEIK to obtainTHFEFENKNKINELKNSFVLSIVEKIQLF dCasJ KYKTIPASVRFLLADYFEEQKLSTKEEAvariants LTIFEEIGKPQNIGFDYIQLKEKDNFTLK shown inKYPLKQAFDYAWENLARLDQNPKANQ bold and FSVDECKRFFKEVFSMEMDNINFKTYAunderlined LLLALKEKTTAFDKKGEGAAKNKSEIIE QIKGWEELDQPFKIIANTLREEVIKKEDELNVLKRQYRETDRKIKTLQNEIKKIKN QIKNLENSKKYSFPEIIKWIDLTEQEQLLDKNKQAKSNYQKAKGDLGLIRGSQKTS INDYFYLTDKVYRKLAQDFGKKMADLREKLLDKNDVNKIKYLSYIVKDNQGYQ YTLLKPLEDKNAEIIELKSEPNGDLKLFEIKSLTSKTLNKFIKNKGAYKEFHSAEFE HKKIKEDWKNYKYNSDFIVKLKKCLSHSDMANTQNWKAFGWDLDKCKSYETIE KEIDQKSYQLVEIKLSKTTIEKWVKENNYLLLPIVNQDITAEKLKVNTNQFTKDW QHIFEKNPNHRLHPEFNIAYRQPTKDYAKEGEKRYSRFQLTGQFMYEYIPQDANY ISRKEQITLFNDKEEQKIQVETFNNQIAK ILNAEDFYVIGID  RGITQLATLCVLNKN GVIQGGFEIFTREFDYTNKQWKHTKLKENRNILDISNLKVETTVNGEKVLVDLSE VKTYLRDENGEPMKNEKGVILTKDNLQKIKLKQLAYDRKLQYKMQHEPELVLSF LDRLENKEQIPNLLASTKLISAYKEGTAYADIDIEQFWNILQTFQTIVDKFGGIENA KKTMEFRQYTELDASFDLKNGVVANMVGVVKFIMEKYNYKTFIAL E DLTFAFG QSIDGINGERLRSTKEDKEVDFKEQENSTLAGLGTYHFFEMQLLKKLSKTQIGNEI KHFVPAFRSTENYEKIVRKDKNVKAKIVSYPFGIVSFVNPRNTSISCPNCKNANK SNRIKKENDRILCKHNIEKTKGNCGFDTANFDENKLRAENKGKNFKYISSGDANA AYNIAVKLLEDKIFEINKK 48 Zinc finger linkerTGEKP peptide 49 SV40 large T PKKKRKV antigen NLS 50 Class IIK(K/R)X(K/R) monopartite NLS consensus 51 Bipartite NLS(K/R)(K/R)X₁₀₋₁₂(K/R)_(3/5) where K/R)3/5 consensus represents at leastthree of either lysine or arginine of five consecutive amino acids 52Class 5 Plant NLS LGKR(K/R)(W/F/Y) 53 (Gly4Ser)n GGGGSn is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 54 Ser(Gly4Ser)n SGGGGSn is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 55 cell-penetratingYGRKKRRQRRR peptide (CPP) 56 cell-penetrating RRQRRTSKLMKR peptide (CPP)57 cell-penetrating GWTLNSAGYLLGKINLKALAALAKKIL peptide (CPP) 58cell-penetrating KALAWEAKLAKALAKALAKHLAKALA peptide (CPP) KALKCEA 59cell-penetrating RQIKIWFQNRRMKWKK peptide (CPP) 60 cell-penetratingYGRKKRRQRRR peptide (CPP) 61 cell-penetrating RKKRRQRR peptide (CPP) 62cell-penetrating YARAAARQARA peptide (CPP) 63 cell-penetratingTHRLPRRRRRR peptide (CPP) 64 cell-penetrating GGRRARRRRRR peptide (CPP)65 ZmWUS2-TALE- TGGCCCTCTGCATCCTCCTCATGATAG 1 binding site CT 66ZmWUS2-TALE- TCTTCCAAATTCCGAAGCGGCCAATG 2 binding site CAAT 67ZmWUS2-TALE- TATCGTATCACCCATGGCCATGACCCC 3 binding site CCT 68ZmODP2-TALE-1 TATTCCTAATATATATATCATACTCTC binding site CAT 69ZmODP2-TALE-2 TCTTCTGGAGTGTACCAGTTGTATAAA binding site TAT 70ZmODP2-TALE-3 TGCACTGTCCAAAATGGCTTCCTGATC binding site CCCT 71Conserved Maize GTACTCATATATGGGCACACATATAG ODP2 promoterACATGTTTTGAGGAAAATGAGACAAA GTATAGTGGAGACTTCCCTAGAAAGCAGAAGAAAAAGAAGTGGTTTATGTTC CGTTAAATCATACTACAACTTTTTTTTATTATACTCTCCATTTTGTCATCATTA GGTACTCATATATGGGCACACATATAGTACTGCCAATTTTTCTTGCTAAAAAA AGTTCCACTATATATATGTATGTATGCACAAATAAACTAATTTTCTTAGAAAA GAAAACCGGTGTAATACATACTAAGGGCTAGTTTGGGAACCCTGGTT 72 ZmWUS2-TALE-msrtrlpsppapspafsadsfsdllrqfdpslfntslfdslppf N terminus of1b N-terminus, gahhteaatgewdevqsglraadappptmrvavtaarppr TALE and RVDakpaprrraaqpsdaspaaqvdlrtlgysqqqqekikpkvr domain in lower RVD, and C-stvaqhhealvghgfthahivalsqhpaalgtvavkyqdmi case; truncated terminalaalpeatheaivgvgkqwsgaralealltvageirgpplqldt TAL terminal domaingqllkiakrggvtaveavhawrnaltgaplnltetltpeqvva domain in upperiasnhggkqaletvqrllpvlcqahgltpeqvvaiasnhggk case andqaletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrl underlinedIpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiasnhggkqaletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgasqltpeqvvaiasnggg RPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNR RIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRR VGVTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSL ERDLDAPSPMHEGDQTRAS 73 ZmWUS2-TALE-MHHHHHHmsrtrlpsppapspafsadsfsdllrqfdps 6xHis tag at N- lb ATFIfntslfdslppfgahhteaatgewdevqsglraadappptm terminus residuesrvavtaarpprakpaprrraaqpsdaspaaqvdlrtlgysqq 2-7 in upper case;qqekikpkvrstvaqhhealvghgfthahivalsqhpaalgt N terminus ofvavkyqdmiaalpeatheaivgvgkqwsgaralealltva TALE, RVDgelrgpplqldtgqllkiakrggvtaveavhawrnaltgapl domain, C-nltetltpeqvvaiasnhggkqaletvqrllpvlcqahgltpe terminus in lowerqvvaiasnhggkqaletvqrllpvlcqahgltpeqvvaiash case; truncateddggkqaletvqrllpvlcqahgltpeqvvaiashdggkqale TAL terminaltvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvl domain in uppercqahgltpeqvvaiasngggkqaletvqrllpvlcqahgltp case andeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaias underlined,ngggkqaletvqrllpvlcqahgltpeqvvaiasnhggkqa extended SV40letvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllp Nuclearvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahglt Localizationpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaia Sequence inshdggkqaletvqrllpvlcqahgltpeqvvaiashdggkq lowercase, doublealetvqrllpvlcqahgltpeqvvaiasngggkqaletvqrll underlined,pvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahg italics; VP64ltpeqvvaiashdggkqaletvqrllpvlcqahgasqltpeq transcriptional vvaiasngggactivation domain RPALESIVAQLSRPDPALAAL is lowercase,TNDHLVALACLGGRPALDAVKKGLPH underlined APALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSR HGLLQLFRRVGVTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQAS LHAFADSLERDLDAPSPMHEGDQTRAS

GRAdalddfdldml ssdalddfdldmlgsdalddfdldmlg sdalddfdldml 74 ZmWUS2-TALE-TGGCCCTCTGCATCCTCCT lb binding site 75 ZmWUS2-TALE-msrtrlpsppapspafsadsfsdllrqfdpslfntslfdslppf N terminus of2b N-terminus, gahhteaatgewdevqsglraadappptmrvavtaarppr TALE and RVDRVD, and C- akpaprrraaqpsdaspaaqvdlrtlgysqqqqekikpkvr domain in lowerterminal domain stvaqhhealvghgfthahivalsqhpaalgtvavkyqdmicase; truncated aalpeatheaivgvgkqwsgaralealltvagelrgpplqldt TAL terminalgqllkiakrggvtaveavhawrnaltgaplnltetltpeqvva domain in upperiasnhggkqaletvqrllpvlcqahgltpeqvvaiasngggk case andqaletvqrllpvlcqahgltpeqvvaiasnhggkqaletvqrl underlinedlpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahltpeqvvaiasniggkqaletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiasnhggkqaletvqrllpvlcqahgasqltpeqvvaiasn gggRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIK RTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQ LFRRVGVTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFA DSLERDLDAPSPMHEGDQTRAS 76 ZmWUS2-TALE-MHHHHHHmsrtrlpsppapspafsadsfsdllrqfdps 6xHis tag at N- 2b ATFlfntslfdslppfgahhteaatgewdevqsglraadappptm terminus residuesrvavtaarpprakpaprrraaqpsdaspaaqvdlrtlgysqq 2-7 in upper case;qqekikpkvrstvaqhhealvghgfthahivalsqhpaalgt N terminus ofvavkyqdmiaaipeatheaivgvgkqwsgaralealltva TALE, RVDgelrgpplqldtgqllkiakrggvtaveavhawrnaltgapl domain, C-nltetltpeqvvaiasnhggkqaletvqrllpvlcqahgltpe terminus in lowerqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiasn case; truncatedhggkqaletvqrllpvlcqahgltpeqvvaiasngggkqale TAL terminaltvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvl domain in uppercqahgltpeqvvaiasniggkqaletvqrllpvlcqahltpeq case andvvaiasniggkqaletvqrllpvlcqahgltpeqvvaiashd underlined,ggkqaletvqrllpvlcqahgltpeqvvaiasngggkqalet extended SV40vqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvlc Nuclearqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpe Localizationqvvaiasniggkqaletvqrllpvlcqahgltpeqvvaiash Sequence indggkqaletvqrllpvlcqahgltpeqvvaiasngggkqale lowercase, doubletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvl underlined,cqahgltpeqvvaiasnhggkqaletvqrllpvlcqahgas italics; VP64ciltDecivvaiasngggRPALESIVAQLSRPDPA transcriptionalLAALTNDHLVALACLGGRPALDAVKK activation domainGLPHAPALIKRTNRRIPERTSHRVADHA is lowercase, QVVRVLGFFQCHSHPAQAFDDAMTQFunderlined GMSRHGLLQLFRRVGVTELEARSGTLP PASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSLERDLDAPSPMHEGD QTRAS

GRAdalddfdldmlgsd alddfdldmlRsdalddfdldmlgsdalddfdldml 77 ZmWUS2-TALE-TGTGTCAACTTCACTTGT 2b binding site 78 ZmWUS2-TALE-msrtrlpsppapspafsadsfsdllrqfdpslfntslfdslppf N terminus of3b N-terminus, gahhteaatgewdevqsglraadappptmrvavtaarppr TALE and RVDRVD, and C- akpaprrraaqpsdaspaaqvdlrtlgysqqqqekikpkvr domain in lowerterminal domain stvaqhhealvghgfthahivalsqhpaalgtvavkyqdmicase; truncated aalpeatheaivgvgkqwsgaralealltvagelrgpplqldt TAL terminalgqllkiakrggvtaveavhawrnaltgaplnltetltpeqvva domain in upperiashdggkqaletvqrllpvlcqahgltpeqvvaiasnhggk case andqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrl underlinedlpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiasnhggkqaletvqrllpvlcqahgltpeqvvaiasnhggkqaletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahgasqltpeqvvaiasnggg RPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNR RIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRR VGVTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSL ERDLDAPSPMHEGDQTRAS 79 ZmWUS2-TALE-MHHHHHHmsrtrlpsppapspafsadsfsdllrqfdps 6xHis tag at N- 3b ATFlfntslfdslppfgahhteaatgewdevqsglraadappptm terminus residuesrvavtaarpprakpaprrraaqpsdaspaaqvdlrtlgysqq 2-7 in upper case;qqekikpkvrstvaqhhealvghgfthahivalsqhpaalgt N terminus ofvavkyqdmiaalpeatheaivgvgkqwsgaraleal1tva TALE, RVDgelrgpplqldtgqllkiakrggvtaveavhawrnaltgapl domain, C-nltetltpeqvvaiashdggkqaletvqrllpvlcqahgltpe terminus in lowerqvvaiasnhggkqaletvqrllpvlcqahgltpeqvvaiasn case; truncatedgggkqaletvqrllpvlcqahgltpeqvvaiasniggkqale TAL terminaltvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvl domain in uppercqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltp case andeqvvaiasniggkqaletvqrllpvlcqahgltpeqvvaias underlined,hdggkqaletvqrllpvlcqahgltpeqvvaiashdggkqa extended SV40letvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllp Nuclearvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahglt Localizationpeqvvaiasngggkqaletvqrl1pvlcqahgltpeqvvaia Sequence insnhggkqaletvqrllpvlcqahgltpeqvvaiasnhggkq lowercase, doublealetvqrllpvlcqahgltpeqvvaiashdggkqaletvqrll underlined,pvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahg italics; VP64ltpeqvvaiasniggkqaletvqrllpvlcqahgasqltpeqv transcriptional vaiasngggactivation domain RPALESIVAQLSRPDPALAAL is lowercase,TNDHLVALACLGGRPALDAVKKGLPH underlined APALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSR HGLLQLFRRVGVTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQAS LHAFADSLERDLDAPSPMHEGDQTRAS

GRAdalddfdldml ssdalddfdldmlgsdalddfdldmlg sdalddfdldml 80 ZmWUS2-TALE-TCGTATCACCCATGGCCAT 3b binding site 81 ZmODP2-TALE-msrtrlpsppapspafsadsfsdllrqfdpslfntslfdslppf N terminus of1b N-terminus, gahhteaatgewdevqsglraadappptmrvavtaarppr TALE and RVDRVD, and C- akpaprrraaqpsdaspaaqvdlrtlgysqqqqekikpkvr domain in lowerterminal domain stvaqhhealvghgfithahivalsqhpaalgtvavkyqdmicase; truncated aalpeatheaivgvgkqwsgaralealltvagelrgpplqldt TAL terminalgqllkiakrggvtaveavhawrnaltgaplnltetltpeqvva domain in upperiashdggkqaletvqrllpvlcqahgltpeqvvaiashdggk case andqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrl underlinedlpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpeqwaiasngggkqaletvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahgasqltpeqvvaias ngggRPALESIVAQLSRPDPALAALTNDHLVALA CLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHS HPAQAFDDAMTQFGMSRHGLLQLFRRVGVTELEARSGTLPPASQRWDRILQAS GMKRAKPSPTSTQTPDQASLHAFADSLERDLDAPSPMHEGDQTRAS 82 ZmODP2-TALE-MHHHHHHmsrtrlpsppapspafsadsfsdllrqfdps 6xHis tag at N- 1b ATFlfntslfdslppfgahhteaatgewdevqsglraadappptm terminusrvavtaarpprakpaprrraaqpsdaspaaqvdlrtlgysqq residuesqqekikpkvrstvaqhhealvghgfthahivalsqhpaalgt 2-7 in upper case;vavkyqdmiaalpeatheaivgvgkqwsgaralealltva N terminus ofgelrgpplqldtgqllkiakrggvtaveavhawrnaltgapl TALE, RVDnltetltpeqvvaiashdggkqaletvqrllpvlcqahgltpe domain, C-qvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiasn terminusgggkqaletvqrllpvlcqahgltpeqvvaiasniggkqale in lowertvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlc case;qahgltpeqvvaiasngggkqaletvqrllpvlcqahgltpe truncatedqvvaiasniggkqaletvqrllpvlcqahgltpeqvvaiasn TAL terminalgggkqaletvqrllpvlcqahgltpeqvvaiasniggkqale domaintvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvl in uppercqahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpe case andqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiasn underlined,iggkqaletvqrllpvlcqahgltpeqvvaiasngggkqale extended SV40tvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvl Nuclearcqahgltpeqvvaiasniggkqaletvqrllpvlcqahgasq Localization ltpeciwaiasngggSequence in RPALESIVAQLSRPDPALAAL lowercase, doubleTNDHLVALACLGGRPALDAVKKGLPH underlined, APALIKRTNRRIPERTSHRVADHAQVVRitalics; VP64 VLGFFQCHSHPAQAFDDAMTQFGMSR transcriptionalHGLLQLFRRVGVTELEARSGTLPPASQR activation domainWDRILQASGMKRAKPSPTSTQTPDQAS is lowercase, LHAFADSLERDLDAPSPMHEGDQTRASunderlined

GRA dalddfdldmlgsdalddfdldmlg sdalddfdldmlgsdalddfdldml 83 ZmODP2-TALE-TCCTAATATATATATCAT 1b binding site 84 ZmODP2-TALE-msrtrlpsppapspafsadsfsdllrqfdpslfntslfdslppf N terminus of2b N-terminus, gahhteaatgewdevqsglraadappptmrvavtaarppr TALE and RVDRVD, and C- akpaprrraaqpsdaspaaqvdlrtlgysqqqqekikpkvr domain in lowerterminal stvaqhhealvghgfthahivalsqhpaalgtvavkyqdmi case; truncateddomain aalpeatheaivgvgkqwsgaralealltvagelrgpplqldt TAL terminalgqllkiakrggvtaveavhawrnaltgaplnltetltpeqvva domain in upperiasnhggkqaletvqrllpvlcqahgltpeqvvaiasngggk case andqaletvqrllpvlcqahgltpeqvvaiasniggkqaletvqrll underlinedpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpeqvvaiasnhggkqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiasnhggkqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahgasqltpeqvvaiasngggRPALESIVAQLSRPDPALAALTNDHLVALA CLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHS HPAQAFDDAMTQFGMSRHGLLQLFRRVGVTELEARSGTLPPASQRWDRILQAS GMKRAKPSPTSTQTPDQASLHAFADSLERDLDAPSPMHEGDQTRAS 85 ZmODP2-TALE-MHHHHHHmsrtrlpsppapspafsadsfsdllrqfdps 6xHis tag at N- 2b ATFlfntslfdslppfgahhteaatgewdevqsglraadappptm terminus residuesrvavtaarpprakpaprrraaqpsdaspaaqvdlrtlgysqq 2-7 in upper case;qqekikpkvrstvaqhhealvghgfthahivalsqhpaalgt N terminus ofvavkyqdmiaalpeatheaivgvgkqwsgaralealltva TALE, RVDgelrgpplqldtgqllkiakrggvtaveavhawrnaltgapl domain, C-nltetltpeqvvaiasnhggkqaletvqrllpvlcqahgltpe terminus in lowerqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiasn case; truncatediggkqaletvqrllpvlcqahgltpeqvvaiashdggkqale TAL terminaltvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvl domain in uppercqahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpe case andqvvaiasnhggkqaletvqrllpvlcqahgltpeqvvaiasn underlined,gggkqaletvqrllpvlcqahgltpeqvvaiasngggkqale extended SV40tvqrllpvlcqahgltpeqvvaiasnhggkqaletvqrllpvl Nuclearcqahgltpeqvvaiasngggkqaletvqrllpvlcqahgltp Localizationeqvvaiasniggkqaletvqrllpvlcqahgltpeqvvaias Sequence inngggkqaletvqrllpvlcqahgltpeqvvaiasniggkqal lowercase, doubleetvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvl underlined,cqahgltpeqvvaiasniggkqaletvqrllpvlcqahgasq italics; VP64 ltpeqvvaiasngggtranscriptional RPALESIVAQLSRPDPALAAL activation domainTNDHLVALACLGGRPALDAVKKGLPH is lowercase, APALIKRTNRRIPERTSHRVADHAQVVRunderlined VLGFFQCHSHPAQAFDDAMTQFGMSR HGLLQLFRRVGVTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQAS LHAFADSLERDLDAPSPMHEGDQTRAS

GRAdalddfdldml ssdalddfdldmlgsdalddfdldmlg sdalddfdldml 86 ZmODP2-TALE-TGTACCAGTTGTATAAAT 2b binding site 87 ZmODP2-T ALE-msrtrlpsppapspafsadsfsdllrqfdpslfntslfdslppf N terminus of3b N-terminus, gahhteaatgewdevqsglraadappptmrvavtaarppr TALE and RVDRVD, and C- akpaprrraaqpsdaspaaqvdlrtlgysqqqqekikpkvr domain in lowerterminal domain stvaqhhealvghgfithahivalsqhpaalgtvavkyqdmicase; truncated aalpeatheaivgvgkqwsgaralealltvagelrgpplqldt TAL terminalgqllkiakrggvtaveavhawmaltgaplnltetltpeqvva domain in upperiasnhggkqaletvqrllpvlcqahgltpeqvvaiasngggk case andqaletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrl underlinedlpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiasnhggkqaletvqrllpvlcqahgltpeqvvaiasnhggkqaletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahgasqltpeqvvaiasngggRPALESIVAQLSRPDPALAALTNDHLVALA CLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHS HPAQAFDDAMTQFGMSRHGLLQLFRRVGVTELEARSGTLPPASQRWDRILQAS GMKRAKPSPTSTQTPDQASLHAFADSLERDLDAPSPMHEGDQTRAS 88 ZmODP2-TALE-MHHHHHHmsrtrlpsppapspafsadsfsdllrqfdps 6xHis tag at N- 3b ATFlfntslfdslppfgahhteaatgewdevqsglraadappptm terminus residuesrvavtaarpprakpaprrraaqpsdaspaaqvdlrtlgysqq 2-7 in upper case;qqekikpkvrstvaqhhealvghgfthahivalsqhpaalgt N terminus ofvavkyqdmiaalpeatheaivgvgkqwsgaralealltva TALE, RVDgelrgpplqldtgqllkiakrggvtaveavhawrnaltgapl domain, C-nltetltpeqvvaiasnhggkqaletvqrllpvlcqahgltpe terminus in lowerqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiash case; truncateddggkqaletvqrllpvlcqahgltpeqvvaiashdggkqale TAL terminaltvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlc domain in upperqahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpe case andqvvaiasniggkqaletvqrllpvlcqahgltpeqvvaiasni underlined, SV40ggkqaletvqrllpvlcqahgltpeqvvaiasngggkqalet Nuclearvqrllpvlcqahgltpeqvvaiasnhggkqaletvqrllpvlc Localizationqahgltpeqvvaiasnhggkqaletvqrllpvlcqahgltpe Sequence inqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiasn lowercase, doublegggkqaletvqrllpvlcqahgltpeqvvaiasngggkqale underlined,tvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvl italics; VP64cqahgltpeqvvaiashdggkqaletvqrllpvlcqahgas transcriptionalqltpeqvvaiasnggg activation domain RPALESIVAQLSRPDPA is lowercase,LAALTNDHLVALACLGGRPALDAVKK underlined GLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQF GMSRHGLLQLFRRVGVTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQT PDQASLHAFADSLERDLDAPSPMHEGD QTRAS

GRAdalddfdldmlgsdalddfdldml gsdalddfdldmlgsdalddfdldml 89 ZmODP2-TALE-TGTCCAAAATGGCTTCCT 3b binding site 90 dCAS9 RNAMHHHHHHmdkkysiglaigtnsvgwa 6xHis tag at N- guided DNAvitdeykvpskkfkvlgntdrhsik terminus residues bindingknligallfdsgetaeatrlkrtar 2-7 in upper case; polypeptide withrrytrrknricylqeifsnemakvd dCas9 domain in Nucleardsffhrleesflveedkkherhpif lowercase; Localization andgnivdevayhekyptiyhlrkklvd extended SV40 Transcriptionstdkadlrliylalahmikfrghfl Nuclear Activator iegdlnpdnsdvdklfiqlvqtynqLocalization sequences with N- lfeenpinasgvdakailsarisks Signal interminal 6xHis rrlenliaqlpgekknglfgnlial lowercase, domainsigltpnfksnfdlaedaklqlskd underlined; VP64 tydddldnllaqigdqyadlflaaktranscriptional nlsdaillsdilrvnteitkaplsa activation domainsmikrydehhqdltllkalvrqqlp in lowercase, ekykeiffdqskngyagyidggasqitalics, and double eefykfikpilekmdgteellvkln underlinedredllrkqrtfdngsiphqihlgel hailrrqedfypflkdnrekiekiltfripyyvgplargnsrfawmtrks eetitpwnfeevvdkgasaqsfiermtnfdknlpnekvlpkhsllyeyft vyneltkvkyvtegmrkpaflsgeqkkaivdllfktnrkvtvkqlkedyf kkiecfdsveisgvedrfnaslgtyhdllkiikdkdfldneenediledi vltltlfedremieerlktyahlfddkvmkqlkmytgwgrlsrklingir dkqsgktildflksdgfanmfmqlihddsltfkediqkaqvsgqgdslhe hianlagspaikkgilqtvkvvdelvkvmgrhkpeniviemarenqttqk gqknsrermkrieegikelgsqilkehpventqlqneklylyylqngrdm yvdqeldinrlsdydvdaivpqsflkddsidnkvltrsdknrgksdnvps eevvkkmknywrqllnaklitqrkfdnltkaergglseldkagfikrqlv etrqitkhvaqildsrmntkydendklirevkvitlksklvsdfrkdfqf ykvreinnyhhahdaylnavvgtalikkypklesefvygdykvydvrkmi akseqeigkatakyffysnimnffkteitlangeirkrplietngetgei vwdkgrdfatvrkvlsmpqvnivkktevqtggfskesilpkrnsdkliar kkdwdpkkyggfdsptvaysvlvvakvekgkskklksvkellgitimers sfeknpidfleakgykevkkdliiklpkyslfelengrkrmlasagelqk gnelalpskyvnflylashyeklkgspedneqkqlfveqhkhyldeiieq isefskrviladanldkvlsaynkhrdkpireqaeniihlftltnlgapa afkyfdttidrkrytstkevldatlihqsitglvetridlsqlggdGSp kkkrkvSSAAGGGGSGRA

91 VPR (VP64-p65- EASGSGRADALDDFDLDMLGSDALDDF Rta) tripartiteDLDMLGSDALDDFDLDMLGSDALDDF transcriptional DLDMLINSRSSGSPKKKRKVGSQYLPDactivator TDDRHRIEEKRKRTYETFKSIMKKSPFS GPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPSGQISQAS ALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQA GEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAP HTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDF SALLGSGSGSRDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWANR PLPASLAPTPTGP VHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKALREMADTVIPQ KEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEILDTFLNDE CLLHAMHISTGLSIFDTSLF 92 Extended SV40ASPKKKRKVEASGS Nuclear Localization Domain 93 ZnFng-WUS1MgPKKKRKVgrlepgekpykcpecgksf SV40 Nuclear ATFsrsdklvrhqrthtgekpykcpecgksfs Localization qssnlvrhqrthtgekpykcpecgksfsrSequence in sddlvrhqrthtgekpykcpecgksfstsg uppercase,slvrhqrthtgekpykcpecgksfsredn underlined; VP64lhthqrthtgekpykcpecgksfsdpgn transcriptional lvrharthtgaaaactivation domain

is lowercase,

double underlined, italics 94 ZnFng-WUS1 ATGGGCCCTAAGAAGAAGCGCAAGGTATF Coding CGGACGCCTTGAGCCCGGCGAAAAGC CATATAAATGCCCAGAGTGTGGCAAGAGCTTCAGCCGCAGCGACAAGCTTGT TCGCCATCAACGCACTCACACCGGCGAGAAGCCTTACAAGTGTCCAGAGTGC GGCAAGAGCTTCAGCCAGAGCAGCAACCTCGTTCGCCATCAAAGGACCCACA CTGGAGAGAAACCATATAAGTGCCCAGAATGCGGAAAAAGCTTCTCCCGCAG CGATGATCTCGTCCGCCACCAGAGGACTCACACCGGAGAGAAGCCTTATAAG TGCCCAGAGTGTGGCAAGTCCTTCTCCACCAGCGGAAGCCTCGTTCGCCATCA GCGCACCCATACTGGAGAAAAACCTTACAAGTGCCCAGAGTGTGGAAAAAGC TTTAGCCGCGAGGACAATCTCCATACCCATCAACGCACCCACACTGGCGAAA AGCCTTACAAATGTCCAGAGTGCGGCAAGTCCTTTTCCGACCCCGGCAACCTT GTTAGGCATCAAAGGACTCATACCGGAGCTGCCGCTGACGCTCTTGACGATTT TGATCTCGACATGCTCGACGCTCTCGATGATTTCGACCTTGACATGCTTGATGC CCTCGACGATTTCGATCTCGATATGCTTGATGCTCTTGACGACTTCGACCTTGA TATGCTCTGA 95 ZnFng-WUS2mgPKKKRKVgrlepgekpvkcpecgks SV40 Nuclear ATF fsdpgalvrhqrthtgekpykcpecgkLocalization sfsrsdnlvrhqrthtgekpykcpecg Sequence inksfsqsgdlrrhqrthtgekpykcpecg uppercase, ksfstsgnlvrhqrthtgekpykcpecgunderlined; VP64 ksfsrsdnlvrhqrthtgekpykcpecg transcriptionalksfsrsdnlvrhqrthtgaaa activation domain

is lowercase,

double underlined, italics 96 ZnFng-WUS2 ATGGGACCAAAAAAGAAGCGCAAGGATF coding TCGGACGCCTTGAGCCCGGCGAAAAG CCATATAAGTGCCCAGAGTGCGGCAAGTCCTTCTCCGATCCCGGCGCCCTCGT TAGGCATCAGAGGACCCACACTGGAGAGAAGCCATACAAGTGTCCAGAGTGC GGAAAAAGCTTCAGCCGCTCCGACAACCTCGTCCGCCATCAGCGCACCCACA CCGGAGAGAAACCATACAAGTGCCCAGAGTGCGGAAAGTCCTTCAGCCAGTC CGGAGATCTTCGCCGCCATCAAAGGACTCATACCGGCGAGAAGCCTTATAAA TGCCCAGAGTGTGGAAAATCCTTCAGCACCAGCGGCAATCTCGTTCGCCACC AGAGGACTCACACTGGCGAGAAGCCATATAAATGTCCAGAATGTGGCAAAAG CTTTTCTCGCTCCGATAACCTCGTTCGCCACCAACGCACCCATACTGGAGAAA AACCTTACAAATGCCCAGAGTGTGGAAAGAGCTTCTCTCGCAGCGACAACCT TGTCCGCCACCAGCGCACTCATACTGGAGCTGCCGCCGACGCCCTCGACGAT TTCGACCTCGATATGCTTGACGCCCTCGATGATTTCGACCTTGATATGCTTGAT GCCCTCGATGACTTCGATCTCGACATGCTCGACGCCCTTGACGACTTTGATCTC GATATGCTCTGA 97 ZnFng-WUS3mgPKKKRKVgrlepgekpykcpecgksf SV40 Nuclear ATFsqlahlrahqrthtgekpykcpecgksf Localization sqsgdlrrhqrthtgekpykcpecgksfSequence in sttgnltvhqrthtgekpykcpecgksf uppercase,sdcrdlarhqrthtgekpykcpecgksf underlined; VP64srsddlvrhqrthtgekpykcpecgksf transcriptional sqssnlvrhqrthtgaaaactivation domain

is lowercase,

double underlined, italics 98 ZnFng-WUS3 ATGGGCCCTAAAAAGAAGCGCAAGGTATF coding CGGACGCCTTGAGCCCGGCGAAAAAC CTTACAAGTGCCCAGAATGCGGAAAATCCTTTAGCCAGCTCGCCCACCTTCGC GCCCATCAGCGCACTCATACCGGAGAGAAGCCATATAAATGCCCAGAGTGTG GAAAGTCCTTCTCCCAGAGCGGCGATCTTCGCCGCCACCAGCGCACCCACAC TGGAGAAAAACCTTATAAGTGTCCAGAATGCGGCAAGAGCTTCAGCACCACC GGCAACCTCACCGTTCACCAGAGGACCCATACCGGCGAGAAGCCATACAAGT GTCCAGAGTGTGGCAAAAGCTTCAGCGACTGCCGCGATCTTGCTCGCCATCA AAGGACTCATACTGGAGAGAAGCCTTACAAGTGTCCAGAGTGCGGCAAGTCC TTCAGCCGCTCCGATGACCTCGTTCGCCATCAGCGCACCCACACCGGCGAAAA GCCATATAAGTGTCCAGAGTGCGGAAAGAGCTTTTCCCAGAGCAGCAACCTT GTTAGGCACCAACGCACCCATACTGGAGCTGCCGCTGATGCTCTCGACGACTT CGACCTCGACATGCTTGACGCTCTCGACGATTTCGATCTTGATATGCTTGATG CCCTCGATGATTTCGATCTCGACATGCTCGATGCTCTCGATGATTTTGACCTTG ACATGCTTTGA 99 ZnFng-WUS4mgPKKKRKVgrlepgekpykcpecgksf SV40 Nuclear ATFsrsdklvrhqrthtgekpykcpecgksf Localization stsgslvrhqrthtgekpykcpecgksfSequence in srednlhthqrthtgekpykcpecgksf uppercase,sqkssliahqrthtgekpykcpecgksf underlined; VP64srsdelvrhqrthtgekpykcpecgksf transcriptional srednlvrhqrthtgaaaactivation domain

is lowercase,

double underlined, italics 100 ZnFng-WUS4 ATGGGACCAAAGAAGAAAAGGAAGGATF coding TCGGCCGCCTTGAGCCCGGCGAAAAG CCTTATAAGTGTCCAGAGTGTGGAAAATCCTTCTCTCGCAGCGATAAGCTCGT TAGGCACCAACGCACCCATACTGGCGAAAAACCATATAAGTGCCCAGAGTGT GGAAAGTCCTTTAGCACTAGCGGCAGCCTTGTTAGGCACCAGCGCACCCACA CCGGCGAAAAGCCTTACAAGTGTCCAGAATGTGGCAAGAGCTTCTCCCGCGA GGATAATCTCCACACTCATCAGCGCACCCATACCGGCGAGAAACCTTACAAG TGTCCAGAATGCGGAAAAAGCTTCAGCCAAAAAAGCAGCCTCATCGCTCATC AGAGGACTCATACCGGAGAGAAGCCTTATAAATGCCCAGAGTGCGGAAAATC CTTCAGCCGCAGCGACGAACTCGTCCGCCATCAACGCACTCACACCGGAGAA AAACCATACAAATGTCCAGAGTGCGGCAAGTCCTTTAGCCGCGAGGACAACC TCCACACCCATCAAAGGACCCACACTGGAGCCGCTGCTGATGCCCTCGACGA CTTCGATCTTGACATGCTTGATGCTCTCGATGATTTCGATCTCGACATGCTTGA CGCCCTCGACGATTTCGACCTCGATATGCTCGACGCCCTTGACGACTTTGACCT TGATATGCTCTGA 101 ZnFng-WUS1-GACTAGGTTGCGGAAGGG binding site in maize WUS2 promoter 102 ZnFng-WUS2-GAGGAGGATGCAGAGGTC binding site (minus strand) in maize WUS2 promoter103 ZnFng-WUS3- GAAGCGGCCAATGCAAGA binding site in maize WUS2 promoter104 ZnFng-WUS4- TAGGTGATATAGGTTGGG binding site (minus strand) in maizeWUS2 promoter 105 ZnFng-WUS1 mggrlepgekpykcpecgksfsrsdklvrhqrthtgekpyBinds SEQ ID AZF DNA kcpecgksfsqssnlvrhqrthtgekpykcpecgksfsrsd NO: 101BINDING dlvrhqrthtgekpykcpecgksfstsgslvrhqrthtgekp DOMAINykcpecgksfsrednlhthqrthtgekpykcpecgksfsdp gnlvrhqrthtgaaa 106 ZnFng-WUS2mggrlepgekpykcpecgksfsdpgalvrhqrthtgekpy Binds SEQ ID AZF DNAkcpecgksfsrsdnlvrhqrthtgekpykcpecgksfsqsg NO: 102 BINDINGdlrrhqrthtgekpykcpecgksfstsgnlvrhqrthtgekp DOMAINykcpecgksfsrsdnlvrhqrthtgekpykcpecgksfsrs dnlvrhqrthtgaaa 107 ZnFng-WUS3mggrlepgekpykcpecgksfsqlahlrahqrthtgekpy Binds SEQ ID AZNDNAkcpecgksfsqsgdlrrhqrthtgekpykcpecgksfsttgn NO: 103 BINDINGltvhqilhtgekpykcpecgksfsdcrdlarhqrthtgekpy DOMAINkcpecgksfsrsddlvrhqrthtgekpykcpecgksfsqss nlvrhqrthtgaaa 108 ZnFng-WUS4mggrlepgekpykcpecgksfsrsdklvrhqrthtgekpy Binds SEQ ID AZF DNAkcpecgksfstsgslvrhqrthtgekpykcpecgksfsredn NO: 104 BINDINGIhthqrthtgekpykcpecgksfsqkssliahqrthtgekpy DOMAINkcpecgksfsrsdelvrhqrthtgekpykcpecgksfsred nlhthqrthtgaaa

The breadth and scope of the present disclosure should not be limited byany of the above-described Examples, but should be defined only inaccordance with the preceding embodiments, the following claims, andtheir equivalents.

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What is claimed is:
 1. A maize plant cell comprising at least oneexogenous gene transcription agent that comprises the polypeptide of SEQID NO:93 and stimulates transcription of the endogenous WUS2 gene,wherein expression of the endogenous WUS2 polypeptide is increased incomparison to the expression of the endogenous WUS2 polypeptide in acontrol maize plant cell, wherein the endogenous WUS2 polypeptide isencoded by an endogenous polynucleotide that is operably linked to anendogenous maize WUS2 promoter of SEQ ID NO:4 or an allelic variantthereof, wherein the at least one exogenous gene transcription agentcomprises an artificial zinc finger (AZF) DNA binding domain polypeptidethat binds to the DNA sequence of SEQ ID NO:101 in the endogenous maizeWUS2 promoter of SEQ ID NO:4, and wherein the maize plant cell can forma regenerable maize plant structure.
 2. The maize plant cell of claim 1,wherein an exogenous polynucleotide encoding a WUS2 polypeptide isabsent before and/or during the increase in expression of the endogenousWUS2 polypeptide.
 3. The maize plant cell of claim 1, wherein theincrease in expression of the endogenous WUS2 polypeptide is sufficientto increase proliferation, somatic embryogenesis, and/or regenerationcapacity of the maize plant cell in comparison to the control maizeplant cell.
 4. The maize plant cell of claim 1, wherein the increase inexpression of the endogenous WUS2 polypeptide is sufficient to increasetransformation efficiency and/or endogenous gene editing efficiency ofthe maize plant cell in comparison to the control maize plant cell. 5.The maize plant cell of claim 1, wherein said cell is located within orobtained from a cultured plant tissue explant, an immature embryo, amature embryo, a leaf, and/or callus or optionally wherein the plantcell is located with or derived from the L1 or L2 layer of the immatureor mature embryo.
 6. The maize plant cell of claim 1, wherein theendogenous WUS2 polypeptide comprises an amino acid sequence having atleast 95%, 96%, 97%, or 99% amino acid sequence identity across theentire length of SEQ ID NO:2.
 7. A method of producing a regenerablemaize plant structure, comprising: (i) introducing into a maize plantcell at least one exogenous gene transcription agent which comprises thepolypeptide of SEQ ID NO:93 and transiently increases expression of anendogenous WUS2 polypeptide, wherein the expression is increased incomparison to the expression of the endogenous WUS2 polypeptide in acontrol maize plant cell, wherein the endogenous WUS2 polypeptide isencoded by an endogenous polynucleotide that is operably linked to anendogenous maize WUS2 promoter of SEQ ID NO:4 or an allelic variantthereof, and wherein the at least one exogenous gene transcription agentcomprises an AZF DNA binding domain polypeptide that binds to the DNAsequence of SEQ ID NO:101 in the endogenous maize WUS2 promoter of SEQID NO:4; and, (ii) culturing the maize plant cell to produce aregenerable maize plant structure.
 8. The method of claim 7, wherein anexogenous polynucleotide encoding a WUS2 polypeptide is absent beforeand/or during the increase in expression of the endogenous WUS2polypeptide.
 9. The method of claim 7, wherein the increase inexpression of the endogenous WUS2 polypeptide is sufficient to increaseproliferation, somatic embryogenesis, and/or regeneration capacity ofthe maize plant cell in comparison to the control maize plant cell. 10.The method of claim 7, wherein the increase in expression of theendogenous WUS2 polypeptide is sufficient to increase transformationefficiency and/or endogenous gene editing efficiency of the maize plantcell in comparison to the control maize plant cell.
 11. The method ofclaim 7, wherein said cell is located within or obtained from a culturedplant tissue explant, an immature embryo, a mature embryo, a leaf,and/or callus or optionally wherein the plant cell is located with orderived from the L1 or L2 layer of the immature or mature embryo. 12.The method of claim 7, wherein the endogenous WUS2 polypeptide comprisesan amino acid sequence having at least 95%, 96%, 97%, or 99% amino acidsequence identity across the entire length of SEQ ID NO:2.